Figure 3 ( B ) Overview of the 8 - oxo - dG : dATP insertion complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . The primer terminal ddC and the incoming dATP are shown in cyan and magenta , respectively . Key residues that form the nascent base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The catalytic metal A and the nucleotide binding metal B are shown in red and neon , respectively . ( C ) 8 - oxo - dG ( green ) establishes a Hoogsteen base pair with dATP ( magenta ) . Asn513 and Arg517 ( purple ) interact with the minor groove of the nascent base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) . ( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , the primer terminus in cyan and the incoming dATP in magenta . A water molecule ( red ) and Mg2 + ( neon ) are occupying the metal A and B sites , respectively .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.(C) 8-oxo-dG (green) establishes a Hoogsteen base pair with dATP (magenta). Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and B sites, respectively.


Figure 4 ( A and B ) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure ( 2PFO , black ) . Binding of 8 - oxo - dG ( green ) in both the anti - ( A ) or syn - ( B ) conformation involves a repositioning of the 5 ′ - phosphate ( red arrow ) . Furthermore , the Arg514 side chain ( purple ) that normally stacks with the templating base and interacts with the 5 ′ - phosphate ( blue - dotted line ) , also repositions ( black arrow ) . In both structures , Arg514 interacts with the C6 - carbonyl of the templating 8 - oxo - dG ( anti ) ( A ) or the C6 - amino group of the incoming dATP ( B ) . The incoming nucleotide is shown in magenta .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A and B) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure (2PFO, black). Binding of 8-oxo-dG (green) in both the anti- (A) or syn-(B) conformation involves a repositioning of the 5′-phosphate (red arrow). Furthermore, the Arg514 side chain (purple) that normally stacks with the templating base and interacts with the 5′-phosphate (blue-dotted line), also repositions (black arrow). In both structures, Arg514 interacts with the C6-carbonyl of the templating 8-oxo-dG(anti) (A) or the C6-amino group of the incoming dATP (B). The incoming nucleotide is shown in magenta.


Figure 5 ( B ) Overview of the 8 - oxo - dG : dC extension complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) and an incoming dUMPNPP ( magenta ) . The primer terminal dC and 8 - oxo - dG are shown in cyan and magenta , respectively . Key residues that form the primer terminal base pair binding pocket ( N513 , R517 and Glu529 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The nucleotide binding metal ( B ) is shown in neon . ( C ) 8 - oxo - dG ( orange ) establishes a Watson - Crick base pair with dC ( cyan ) at the primer terminus . Tyr505 , Arg517 , and Glu529 ( purple ) interact with the minor groove of the base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) . ( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , incoming dUMPNPP in magenta , and primer terminal dC in cyan . Expectedly , Mg2 + ( neon ) is occupying the metal B site . However , the metal A is absent in the structure , resulting in a non - catalytic position of the 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dC and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513, R517 and Glu529) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.(C) 8-oxo-dG (orange) establishes a Watson-Crick base pair with dC (cyan) at the primer terminus. Tyr505, Arg517, and Glu529 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dC in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


Figure 6 ( B ) Overview of the 8 - oxo - dG : dC extension complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) and an incoming dUMPNPP ( magenta ) . The primer terminal dA and 8 - oxo - dG are shown in cyan and magenta , respectively . Key residues that form the primer terminal base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The nucleotide binding metal ( B ) is shown in neon . ( C ) 8 - oxo - dG ( orange ) establishes a Hoogsteen base pair with dA ( cyan ) at the primer terminus . Tyr505 and Arg517 ( purple ) interact with the minor groove of the base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) . ( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , incoming dUMPNPP in magenta , and primer terminal dA in cyan . Expectedly , Mg2 + ( neon ) is occupying the metal B site . However , the metal A is absent in the structure , resulting in a non - catalytic position of the 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dA and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.(C) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


Figure 7 ( A ) Glu529 ( purple ) forms a hydrogen bond with the C2 - amino group of the primer terminal templating 8 - oxo - dG ( orange ) in the anti - conformation . ( B ) This interaction is abolished when 8 - oxo - dG ( orange ) adopts the syn - conformation . An overlay with the 8 - oxo - dG ( anti ) - containing structure reveals that Glu529 ( black ) would likely clash ( red - dotted line ) with the C8 - carbonyl of 8 - oxo - dG ( syn ) . Repositioning of Glu529 ( purple ) places the side chain 4 . 5 Å away from the C8 - carbonyl . ( C ) Comparison of catalytic efficiencies for extension past an 8 - oxo - dG ( anti ) : dC , 8 - oxo - dG ( syn ) : dA and dG : dC base pairs at the primer terminus using WT and E529A Pol λ .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.


Figure 8 ( A ) Undamaged structure containing a templating dG at the primer terminus ( 2PFO ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at the primer terminus . ( B ) Undamaged structure containing a templating dA at the primer terminus ( 1XSN ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at the primer terminus .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Undamaged structure containing a templating dG at the primer terminus (2PFO) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus. (B) Undamaged structure containing a templating dA at the primer terminus (1XSN) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus.


Figure 1 ( A ) Gross morphology of control ( Ctrl ) , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , and Sas - 4 − / − Usp28 − / − embryos at E9 . 5 . At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text . Scale bar = 500 μm . ( B , C ) Immunostaining for p53 ( B ) and Cleaved - Caspase3 ( Cl - CASP3 , C ) on transverse sections of Ctrl , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , and Sas - 4 − / − Usp28 − / − embryos at E9 . 5 . The area shown encompasses the ventral neural tube and surrounding mesenchyme . Asterisks in ( B ) denote non - specific staining of blood cells . Scale bar = 50 μm . ( D , E ) Quantifications of the p53 ( D ) and Cl - CASP3 ( E ) fluorescence signals shown in ( B ) and ( C ) , respectively . Three embryos per genotype were used for the quantifications . * * * p < 0 . 001 , * p < 0 . 05 ( two - tailed student ' s T - test , one - way ANOVA with Tukey ' s multiple comparisons tests gave similar results ) . Bars represent mean ± s . d . ( D ) Ctrl : 1 . 00 ± 0 . 06 ( n = 1770 cells ) ; Sas - 4 − / − : 6 . 84 ± 0 . 34 ( n = 275 ) , Sas - 4 − / − 53bp1 − / − : 1 . 85 ± 0 . 75 ( n = 690 ) , Sas - 4 − / − Usp28 − / − : 1 . 50 ± 0 . 48 ( n = 975 ) . ( E ) Ctrl : 1 . 00 ± 0 . 70 ( n = 1690 ) ; Sas - 4 − / − : 7 . 88 ± 2 . 62 ( n = 175 ) ; Sas - 4 − / − 53bp1 − / − : 1 . 43 ± 0 . 71 ( n = 1030 ) ; Sas - 4 − / − Usp28 − / − : 1 . 57 ± 0 . 44 ( n = 950 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Gross morphology of control (Ctrl), Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm.(B, C) Immunostaining for p53 (B) and Cleaved-Caspase3 (Cl-CASP3, C) on transverse sections of Ctrl, Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non-specific staining of blood cells. Scale bar = 50 μm. (D, E) Quantifications of the p53 (D) and Cl-CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. *** p < 0.001, *p<0.05 (two-tailed student's T-test, one-way ANOVA with Tukey's multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n=1770 cells); Sas-4−/−: 6.84 ± 0.34 (n=275), Sas-4−/− 53bp1−/−: 1.85 ± 0.75 (n=690), Sas-4−/− Usp28−/−: 1.50 ± 0.48 (n=975). (E) Ctrl: 1.00 ± 0.70 (n=1690); Sas-4−/−: 7.88 ± 2.62 (n=175); Sas-4−/− 53bp1−/−: 1.43 ± 0.71 (n=1030); Sas-4−/− Usp28−/−: 1.57 ± 0.44 (n=950).


Figure 2 ( A , B ) Whole - mount immunostaining and quantification for p53 on Ctrl and Sas - 4 − / − embryos at E7 . 5 ( A ) and E6 . 5 ( B ) . Representative sagittal planes are shown and the dotted lines demarcate the epiblast . Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below , normalized to control embryos in the same batch at E7 . 5 ( 4 embryos ) and at E6 . 5 ( 9 embryos ) . * * * p < 0 . 001 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bars = 100 μm . ( A ) Ctrl : 1 . 04 ± 0 . 00 ( n = 495 cells ) ; Sas - 4 - / - : 1 . 65 ± 0 . 19 ( n = 850 ) . ( B ) Ctrl : 1 . 00 ± 0 . 18 ( n = 1665 ) ; Sas - 4 - / - : 1 . 16 ± 0 . 44 ( n = 1330 ) ( C , D ) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6 . 5 ( C ) or sagittal planes of whole - mount immunostaining at E5 . 5 ( D ) . Quantification of the fluorescent signals in the epiblast ( Ep ) versus visceral endoderm ( VE ) are shown below . Four embryos per genotype were used for the quantifications . * * * * p < 0 . 0001 , * * * p < 0 . 001 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bar = 50 μm . ( C ) 53BP1 in Ep : 1 . 00 ± 0 . 10 ( n = 225 cells ) ; 53BP1 in VE : 0 . 37 ± 0 . 06 ( n = 105 ) ; USP28 in Ep : 1 . 00 ± 0 . 08 ( n = 235 ) ; USP28 in VE : 0 . 17 ± 0 . 05 ( n = 90 ) . ( D ) 53BP1 in Ep : 1 . 00 ± 0 . 24 ( n = 225 ) ; 53BP1 in VE : 0 . 68 ± 0 . 14 ( n = 115 ) ; USP28 in Ep : 1 . 00 ± 0 . 23 ( n = 280 ) ; USP28 in VE : 0 . 28 ± 0 . 00 ( n = 110 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A, B) Whole-mount immunostaining and quantification for p53 on Ctrl and Sas-4−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). *** p < 0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n=495 cells); Sas-4-/-: 1.65 ± 0.19 (n=850). (B) Ctrl: 1.00 ± 0.18 (n=1665); Sas-4-/-: 1.16 ± 0.44 (n=1330)(C, D) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6.5 (C) or sagittal planes of whole-mount immunostaining at E5.5 (D). Quantification of the fluorescent signals in the epiblast (Ep) versus visceral endoderm (VE) are shown below. Four embryos per genotype were used for the quantifications. **** p < 0.0001, ***p<0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 50 μm. (C) 53BP1 in Ep: 1.00 ± 0.10 (n=225 cells); 53BP1 in VE: 0.37 ± 0.06 (n=105); USP28 in Ep: 1.00 ± 0.08 (n=235); USP28 in VE: 0.17 ± 0.05 (n=90). (D) 53BP1 in Ep: 1.00 ± 0.24 (n=225); 53BP1 in VE: 0.68 ± 0.14 (n=115); USP28 in Ep: 1.00 ± 0.23 (n=280); USP28 in VE: 0.28 ± 0.00 (n=110).


Figure 3 ( A ) Immunostaining for the centrosome marker γ - tubulin ( TUBG ) on Ctrl and Sas - 4 − / − primary mESCs . The bottom panels are magnifications of the areas marked in the top panels . Scale bars = 20 μm ( top ) and 10 μm ( bottom ) . ( B ) Immunostaining and quantification for p53 on Ctrl and Sas - 4 − / − primary mESCs in pluripotent and partially differentiated ( diff . ) conditions . The quantification of p53 fluorescence intensities was normalized to Ctrl ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - tailed student ' s T - test , the comparison of the genotypes in the " Pluripotent " condition was not significant using the one - way ANOVA with Tukey ' s multiple comparisons tests ) . Bars represent mean ± s . d . Scale bar = 50 μm . Pluripotent Ctrl : 1 . 00 ± 0 . 02 ( n = 2030 cells ) ; pluripotent Sas - 4 - / - : 1 . 17 ± 0 . 10 ( n = 2170 ) ; partially diff . Ctrl : 0 . 62 ± 0 . 13 ( n = 2745 ) ; partially diff . Sas - 4 - / - : 1 . 12 ± 0 . 24 ( n = 2000 ) . ( C , D ) Three - day growth curves of WT and Sas - 4 − / − ( C ) or p53 − / − and Sas - 4 − / − p53 − / − ( D ) primary mESCs in the indicated conditions starting with 105 cells on Day 0 ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . For details of the measurements , see Materials and Methods ( Tables 3 and 4 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Immunostaining for the centrosome marker γ-tubulin (TUBG) on Ctrl and Sas-4−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom).(B) Immunostaining and quantification for p53 on Ctrl and Sas-4−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test, the comparison of the genotypes in the "Pluripotent" condition was not significant using the one-way ANOVA with Tukey's multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n=2030 cells); pluripotent Sas-4-/-: 1.17 ± 0.10 (n=2170); partially diff. Ctrl: 0.62 ± 0.13 (n=2745); partially diff. Sas-4-/-: 1.12 ± 0.24 (n=2000).(C, D) Three-day growth curves of WT and Sas-4−/− (C) or p53−/− and Sas-4−/− p53−/− (D) primary mESCs in the indicated conditions starting with 105 cells on Day 0 (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).


Figure 4 ( A ) Sagittal planes of whole - mount immunostaining and quantification for TUBG on WT mouse embryos from E3 . 5 to E6 . 5 . The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass ( E3 . 5 ) or epiblast ( E5 . 5 and E6 . 5 ) . Bars represent mean ± s . d ( two - tailed student ' s T - test or one - way ANOVA with Tukey ' s multiple comparisons tests ) . Scale bars : E3 . 5 and E5 . 5 = 15 μm ; E6 . 5 = 30 μm and 3 μm ( insets ) . E3 . 5 : 88 ± 26 % ( n = 73 cells from 5 embryos ) ; E5 . 5 : 92 ± 10 % ( n = 270 from 3 embryos ) ; E6 . 5 : 79 ± 12 % ( n = 12 , 082 from 3 embryos ) . ( B , C ) Representative gSTED super - resolution images of immunostaining for TUBG and either CEP164 ( B ) or ODF2 ( C ) . Whole - mount staining was used for the CEP164 data as well as for the E3 . 5 blastocysts for ODF2 , whereas staining on sections was used for ODF2 at E5 . 5 and E6 . 5 . Quantification of the percentage of centrosomes ( TUBG ) with CEP164 or ODF2 are shown on the right . * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p < 0 . 05 ( two - tailed student ' s T - test or one - way ANOVA with Tukey ' s multiple comparisons tests ; the ODF2 data between E5 . 5 and E6 . 5 was not significant using the latter test ) . Bars represent mean ± s . d . Scale bar = 0 . 5 μm . CEP164 at E3 . 5 : 2 ± 2 % , ( n = 92 centrosomes from 4 embryos ) ; E5 . 5 : 50 ± 2 % ( n = 249 from 3 embryos ) ; E6 . 5 : 62 ± 0 % ( n = 856 from 3 embryos ) . ODF2 at E3 . 5 : 20 ± 9 % ( n = 108 from 5 embryos ) ; E5 . 5 : 51 ± 4 % ( n = 294 from 5 embryos ) ; E6 . 5 : 60 ± 2 % ( n = 1 , 183 from 4 embryos ) . ( D ) Immunostaining and quantification of the area of TUBG and PCNT signals from E5 . 5 and E6 . 5 centrosomes in epiblast cells . The separate channels for the marked centriole are shown in the insets . * * * p < 0 . 001 , * * p < 0 . 01 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bar = 2 μm . Quantification of PCNT at E5 . 5 : 0 . 22 ± 0 . 01 μm ( n = 103 centrosomes from 3 embryos ) ; E6 . 5 : 0 . 31 ± 0 . 02 μm ( n = 342 from 3 embryos ) . Quantification of TUBG at E5 . 5 : 0 . 15 ± 0 . 02 μm ( n = 108 from 4 embryos ) ; E6 . 5 : 0 . 25 ± 0 . 01 μm ( n = 311 from 3 embryos ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Sagittal planes of whole-mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n=73 cells from 5 embryos); E5.5: 92 ± 10% (n=270 from 3 embryos); E6.5: 79 ± 12% (n=12,082 from 3 embryos).(B, C) Representative gSTED super-resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole-mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. **** p < 0.0001, *** p < 0.001, * p < 0.05 (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n=92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n=249 from 3 embryos); E6.5: 62 ± 0% (n=856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n=108 from 5 embryos); E5.5: 51 ± 4% (n=294 from 5 embryos); E6.5: 60 ± 2% (n=1,183 from 4 embryos).(D) Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. *** p < 0.001, ** p < 0.01 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n=103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n=342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n=108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n=311 from 3 embryos).


Figure 1MEFs expressing vector , TRPML - 1 , or GRP1 - DD were treated with BSA or palmitate ( 250 μM ) for 3 h and stained as in ( A ) . aspect ratio ( O ) , branch length ( P ) , and roundness ( Q ) of mitochondria in the cells in ( N ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).


Figure 2TMRE ( 100 nM ) and MitoTracker green ( 200 nM ) staining in MEFs treated with BSA or palmitate ( 250 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . TMRE intensity in ( A ) on a per cell basis . MitoSOX ( 5 μM ) and MitoTracker Green ( 200 nM ) staining in MEFs treated with vehicle ( DMSO ) or C2 - ceramide ( 50 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . MitoSOX intensity for ( C ) on a per cell basis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.


Figure 3NAD ( P ) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in ( A ) 4 - 8 h after treatment with vehicle ( n = 5 ) or 120 mg / kg SH - BC - 893 ( n = 4 ) by gavage at ZT8 . 5 . Mice were sacrificed and imaged in pairs ( SD and HFD ) . Citrate synthase staining in the arcuate nucleus of mice in ( E ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 3 . imaging was performed in the areas of the

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O

Figure 3NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.imaging was performed in the areas of the cortex


Figure 4NAD ( P ) H autofluorescence was evaluated in epididymal fat ( eWAT ) freshly resected from the mice in ( A ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 5 . NAD ( P ) H intensity from ( B ) calculated on a per cell basis ; 16 - 25 cells from each of the 5 mice per group were quantified ( 87 - 109 total cells per treatment group ) . TMRE ( 100 nM ) and MitoTracker Green staining ( 200 nM ) in eWAT resected from mice in ( D ) treated as in ( B , C ) ; n = 5 . TMRE ( F ) or MitoTracker ( G ) intensity for ( E ) calculated on a per cell basis ; 15 - 24 cells from each of the 5 mice in each group were quantified ( 99 - 104 total cells per treatment group ) . MitoSOX ( 5 μM ) and MitoTracker Green staining in eWAT resected from the mice in ( D ) treated as in ( B and E ) ; n = 5 . MitoSOX ( I ) or MitoTracker ( J ) intensity calculated as in ( F and G ) ; 10 - 13 cells from each of the 5 mice in each group were quantified ( 57 - 58 total cells per treatment group ) . Chop , Xbp1s , Atf6 , and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg / kg SH - BC - 893 on Monday , Wednesday , and Friday for the final 3 weeks ; n = 7 - 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.


Figure 5Plasma adiponectin ( A ) or leptin ( B ) in mice maintained on a SD or HFD for 8 - 9 weeks then gavaged with vehicle or 120 mg / kg SH - BC - 893 4 h prior to blood collection . Pair - fed mice were provided with the amount of food consumed by SH - BC - 893 - treated mice in 4 h but left untreated ; n = 8 - 10 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.


Figure 6Average daily distance run by HFD - fed mice treated with vehicle ( n = 8 ) or SH - BC - 893 ( 120 mg / kg , n = 6 ) on Mondays , Wednesdays , and Fridays over 4 weeks when housed with a running wheel . Body weight change in the mice in ( O , P ) over the 24 h after the first treatment ( Q ) or after the 3 week course of treatment ( R ) ; n = 9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.Body weight change in the mice in (O,P) over the 24 h after the first treatment (Q) or after the 3 week course of treatment (R); n=9.


Figure 7Representative H & E stained liver sections from the mice shown in Fig . 6F after 4 weeks of treatment with vehicle or 120 mg / kg SH - BC - 893 . Scale bar , 10 μm . Blood glucose levels ( F ) or area under the curve ( AUC , ( G ) ) during an oral glucose tolerance test performed in the same mice as in ( E ) . Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH - BC - 893 from the mice in Fig . 5D , E or in 9 week old SD - fed mice treated with vehicle ( same mice as in Fig . 5J , K , 0 weeks on HFD ) ; n = 8 . Insulin levels in the plasma collected 4 h after treatment with vehicle or SH - BC - 893 from the same mice as in Fig . 5A , B ; n = 8 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.


Figure 1 ( B ) Flow cytometry analysis of WT to Il21r - / - splenic SWHEL B cells over time . ( C ) WT to Il21r - / - SWHEL B cell ratio within CTV division peaks . Data information : Data in ( B ) is representative of 8 - 9 biological replicates ( n = 8 - 9 ) pooled from two independent experiments with statistical analysis by one - way ANOVA with Tukey ' s post - test . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 . Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments . ( E ) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks . Data information : Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Flow cytometry analysis of WT to Il21r-/- splenic SWHEL B cells over time. (C) WT to Il21r-/- SWHEL B cell ratio within CTV division peaks. Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.(E) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks. Data information: Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


Figure 2 ( B ) Flow cytometry analysis showing WT to Il21r - / - SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 . ( D ) Ratio of the proportion of BrdU positive WT and Il21r - / - SWHEL B cells with 2N DNA content 4 , 10 and 12 hours post BrdU pulse on day 3 . 5 post immunization . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Flow cytometry analysis showing WT to Il21r-/- SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.(D) Ratio of the proportion of BrdU positive WT and Il21r-/- SWHEL B cells with 2N DNA content 4, 10 and 12 hours post BrdU pulse on day 3.5 post immunization. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


Figure 3 ( C ) Rate of de novo cell cycle entry 10 hours post in vivo IL - 21 ( 2 µg ) or saline pulse . The ratio of WT to Il21r - / - cells not in S / G2 at the time of pulse ( BrdU - cells ) and containing > 2N DNA content 10 hours after pulse is shown . Data information : Data are representative of 10 - 12 biological replicates ( n = 10 - 12 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C) Rate of de novo cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA content 10 hours after pulse is shown. Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.


Figure 4Phosphoflow analysis of naïve WT or Il21r - / - B cells following in vitro culture for 3 hours with or without IL - 21 ( 20 ng / mL ) and / or in the presence of BCR cross - linking ( biotinylated anti - Igκ + anti - Igλ and avidin - mediated cross - linking ) or agonistic anti - CD40 . ( A ) Exemplary p - AKT ( S473 ) staining and ( B ) quantification of p - AKT median fluorescence intensity ( MedFI ) . ( C ) Exemplary p - S6 ( Ser235 / 236 ) staining and ( D ) quantification of frequency of p - S6 positive cells Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( D ) , t test ( B , E ) * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ( F , In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( F ) Exemplary CTV division peaks . G ) In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( G ) Quantification of the proportion SWHEL B that had divided and thus diluted CTV . Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by two - way ANOVA with correction for multiple comparison using Šidák method ( G ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Phosphoflow analysis of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR cross-linking (biotinylated anti-Igκ + anti-Igλ and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT (S473) staining and (B) quantification of p-AKT median fluorescence intensity (MedFI). (C) Exemplary p-S6 (Ser235/236) staining and (D) quantification of frequency of p-S6 positive cells Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.(F, In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (F) Exemplary CTV division peaks.G) In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (G) Quantification of the proportion SWHEL B that had divided and thus diluted CTV. Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


Figure 5 ( B , C ) Analysis of splenic OTII Tfh cells by flow cytometry . ( B ) Tfh cell count and ( C ) Proportion of Il21Gfp / + OTII Tfh cells transcribing the Il21 locus and thus expressing GFP . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( B , C ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . Analysis of splenic WT and Il21r - / - SWHEL B cells by flow cytometry . ( D ) CTV profile of and ( E ) absolute cell count in pooled CTV division peaks ( 0 - 4 , 5 - 8 , 9 - 11 + ) . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . D , show concatenated data representative of 3 - 5 biological replicates . Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm - Šídák method . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . ( F ) Cell surface expression ( median fluorescence intensity , MedFI ) of CD38 , IgD and FAS across individual CTV division peaks . ( G ) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . F and G show concatenated data representative of 3 - 5 biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B, C) Analysis of splenic OTII Tfh cells by flow cytometry. (B) Tfh cell count and (C) Proportion of Il21Gfp/+ OTII Tfh cells transcribing the Il21 locus and thus expressing GFP. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. Statistical analysis by one-way ANOVA with Tukey's post-test (B, C). * p ≤ 0.05; ** p ≤ 0.01.Analysis of splenic WT and Il21r-/- SWHEL B cells by flow cytometry. (D) CTV profile of and (E) absolute cell count in pooled CTV division peaks (0-4, 5-8, 9-11+). Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, show concatenated data representative of 3-5 biological replicates. Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.(F) Cell surface expression (median fluorescence intensity, MedFI) of CD38, IgD and FAS across individual CTV division peaks. (G) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. F and G show concatenated data representative of 3-5 biological replicates.


Figure 6 ( A - E ) Analysis of splenic SWHEL B cells and PC by flow cytometry . ( A ) Proportion of SWHEL B cells with a FAS + GL7 + GC phenotype , ( B ) SWHEL GC B cell count and ( C ) representation of IL - 21R deficient cells among total SWHEL GC B cells . ( D ) CD98 + CD138 + _ SWHEL PC count and ( E ) representation of IL - 21R deficient cells among total SWHEL PC . Data information : Data are representative of 6 - 10 biological replicates ( n = 6 - 10 ) from two independent experiments . Data in B , D were analyzed after log transformation with with statistical analysis by Multiple paired t tests ( A , B , D ) and p - values corrected for multiple comparisons using Holm - Šídák method or One - way ANOVA with Tukey ' s post - test ( C ) or t test ( E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-E) Analysis of splenic SWHEL B cells and PC by flow cytometry. (A) Proportion of SWHEL B cells with a FAS+GL7+ GC phenotype, (B) SWHEL GC B cell count and (C) representation of IL-21R deficient cells among total SWHEL GC B cells. (D) CD98+ CD138+_SWHEL PC count and (E) representation of IL-21R deficient cells among total SWHEL PC. Data information: Data are representative of 6-10 biological replicates (n = 6-10) from two independent experiments. Data in B, D were analyzed after log transformation with with statistical analysis by Multiple paired t tests (A, B, D) and p-values corrected for multiple comparisons using Holm-Šídák method or One-way ANOVA with Tukey's post-test (C) or t test (E).


Figure 1Western blotting in the WT , Trim71 knockout ( Trim71 _ KO ) , and the Trim71 mutant ( R783H ) mESCs . The signals from the pluripotency factors Nanog and Oct4 were obtained through short ( ~ 10 seconds ) exposure . Colony formation assay for mESCs . The mESCs were cultured in 15 % FBS + Lif for 7 days , and the resultant colonies were fixed and stained for AP . Exit pluripotency assay for mESCs . The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i + Lif medium for 5 days . The resultant colonies were fixed and stained for AP . Western blotting of pluripotency factors during EB formation . The signals from the pluripotency factors Nanog and Oct4 were obtained through long ( 5 - 10min ) exposure . Immunofluorescence ( IF ) staining showing the expression level of Rex1 in mESCs cultured in the stemness ( 2i + Lif ) and differentiating ( 15 % FBS - Lif ) conditions . Relative intensity of IF signals from individual cells in experiment associated with E . The results represent the means ( ± SD ) of three independent IF stainings . Expression of neural lineage markers during mESCs neural differentiation . Expression of neural lineage markers during mESCs neural differentiation . IF staining of Nestin . The quantifications represent the means ( ± SD ) of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O B-PANEL_START O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 1Western blotting in the WT, Trim71 knockout (Trim71_KO), and the Trim71 mutant (R783H) mESCs. The signals from the pluripotency factors Nanog and Oct4 were obtained through short (~10 seconds) exposure.Colony formation assay for mESCs. The mESCs were cultured in 15%FBS + Lif for 7 days, and the resultant colonies were fixed and stained for AP.Exit pluripotency assay for mESCs. The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i+Lif medium for 5 days. The resultant colonies were fixed and stained for AP.Western blotting of pluripotency factors during EB formation. The signals from the pluripotency factors Nanog and Oct4 were obtained through long (5-10min) exposure.Immunofluorescence (IF) staining showing the expression level of Rex1 in mESCs cultured in the stemness (2i + Lif) and differentiating (15% FBS - Lif) conditions.Relative intensity of IF signals from individual cells in experiment associated with E. The results represent the means (± SD) of three independent IF stainings.Expression of neural lineage markers during mESCs neural differentiation.Expression of neural lineage markers during mESCs neural differentiation.IF staining of Nestin. The quantifications represent the means (± SD) of three independent experiments.


Figure 2Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome . In each CLIP - seq , there are two biological replicates . The binding regions present in both of the biological replicates are used for the analysis . CLIP - qRT - PCR for the identified target mRNAs of the Trim71 mutant R783H . The results represent the means ( ± SD ) of three independent experiments . UCSC genome browser snapshot for the CLIP - seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus . The red box indicates the binding region of the Trim71 mutant R783H . The inputs are from the size - matched input samples in the CLIP - seq analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome. In each CLIP-seq, there are two biological replicates. The binding regions present in both of the biological replicates are used for the analysis.CLIP-qRT-PCR for the identified target mRNAs of the Trim71 mutant R783H. The results represent the means (±SD) of three independent experiments.UCSC genome browser snapshot for the CLIP-seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus. The red box indicates the binding region of the Trim71 mutant R783H. The inputs are from the size-matched input samples in the CLIP-seq analysis.


Figure 3B . A representative set of Western blots in the WT and the R783H Trim71 mESCs . C . Quantification of the target proteins in the WT and the R783H Trim71 mESCs . Data information : The results represent the means ( ± SD ) of three independent experiments . One - Way ANOVA was used to determine the significance of the difference , * p < 0 . 05 . The Western blots listed in B is the Biological Replicate

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. A representative set of Western blots in the WT and the R783H Trim71 mESCs. C. Quantification of the target proteins in the WT and the R783H Trim71 mESCs. Data information: The results represent the means (± SD) of three independent experiments. One-Way ANOVA was used to determine the significance of the difference, *p<0.05. The Western blots listed in B is the Biological Replicate 1


Figure 4Western blotting in the WT and the Trim71 ( R783H ) mESCs . Quantification of Lsd1 protein and mRNA levels . Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications , respectively . Polysome analysis in the WT and the Trim71 ( R783H ) mESCs . Quantification of the indicated mRNA distribution in the RNP , 80S , and polysome fractions from the WT and the Trim71 ( R783H ) mESCs . Western blotting in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) . Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) . Polysome analysis in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) . Quantification of the indicated mRNA distribution in the RNP , 80S , and polysome fractions from the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Western blotting in the WT and the Trim71 (R783H) mESCs.Quantification of Lsd1 protein and mRNA levels. Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications, respectively.Polysome analysis in the WT and the Trim71 (R783H) mESCs.Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT and the Trim71 (R783H) mESCs.Western blotting in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).Polysome analysis in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H). Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Figure 5Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA ' s 3 ' UTR . CLIP - RIP followed by qRT - PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . The mRNA signals from the E14 mESCs were set as 1 for relative comparison . Western blotting in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . Quantification of Lsd1 protein and mRNA in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . Beta - Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification , respectively . Exit pluripotency assay for mESCs . Representative Western blotting and quantification of pluripotency factors during EB formation . Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O

Figure 5Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA's 3'UTR.CLIP-RIP followed by qRT-PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. The mRNA signals from the E14 mESCs were set as 1 for relative comparison.Western blotting in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs.Quantification of Lsd1 protein and mRNA in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. Beta-Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification, respectively.Exit pluripotency assay for mESCs.Representative Western blotting and quantification of pluripotency factors during EB formation.Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.


Figure 6Western blotting in the WT and the Trim71 ( R783H ) mESCs with dox - inducible expression of Lsd1 - GFP . Exit pluripotency assay for mESCs . Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs . Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O

Figure 6Western blotting in the WT and the Trim71(R783H) mESCs with dox-inducible expression of Lsd1-GFP.Exit pluripotency assay for mESCs.Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs.Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.


Figure 7Representative Western blotting and quantification in the WT and the R783H / + mESCs . Exit pluripotency assay for the WT and the R783H / + mESCs . Representative Western blotting and quantification of pluripotency factors in differentiating mESCs . Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation . Western blotting in the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP . Exit pluripotency assay for the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP . Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP . Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative Western blotting and quantification in the WT and the R783H/+ mESCs.Exit pluripotency assay for the WT and the R783H/+ mESCs.Representative Western blotting and quantification of pluripotency factors in differentiating mESCs.Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.Western blotting in the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.Exit pluripotency assay for the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Figure 1 ( C ) Electrophoretic mobility - shift assay ( EMSA ) with biotin - labeled RORA consensus , rs2978980T or rs2978980G probes and HGC - 27 or MGC80 - 3 nuclear extracts . HGC - 27 or MGC80 - 3 left panel : EMSA with RORA consensus or rs2978980T probes . Lane 1 - 8 : from the first lane at the left side to the right side . Lanes 4 and 8 , probe only ; lanes 2 and 6 , probe and nuclear extracts ; lanes 1 , 3 , 5 and 7 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 1 and 5 ) or RORA consensus probes ( lanes 3 and 7 ) . HGC - 27 or MGC80 - 3 right panel : EMSA with rs2978980T or rs2978980G probes . Lanes 1 and 6 , probe only ; lanes 2 and 7 , probe and nuclear extracts ; lanes 3 - 5 and 8 - 10 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 5 and 8 ) , rs2978980G ( lanes 3 and 10 ) or RORA consensus probes ( lanes 4 and 9 ) . ( F ) Suppression of RORA using siRNAs ( siRORA - 1 and siRORA - 2 ) could markedly down - regulate lncPSCA expression in MGC80 - 3 cells but not in HGC - 27 cells . In detail , MGC80 - 3 and HGC - 27 cells were transfected with siRORA - 1 and siRORA - 2 , lncPSCA and RORA expression levels were measured by RT - qPCR and Western Blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1  (C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).   (F) Suppression of RORA using siRNAs (siRORA-1 and siRORA-2) could markedly down-regulate lncPSCA expression in MGC80-3 cells but not in HGC-27 cells. In detail, MGC80-3 and HGC-27 cells were transfected with siRORA-1 and siRORA-2, lncPSCA and RORA expression levels were measured by RT-qPCR and Western Blot.


Figure 2 ( E ) lncPSCA - knockout significantly promoted growth of MGC80 - 3 ( n = 8 ) or HGC - 27 ( n = 7 ) xenografts compared with control xenografts after 27 or 18 days . ( F ) HE and Ki67 staining of MGC80 - 3 or HGC - 27 xenografts . Scale bar = 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (E) lncPSCA-knockout significantly promoted growth of MGC80-3 (n = 8) or HGC-27 (n = 7) xenografts compared with control xenografts after 27 or 18 days.  (F) HE and Ki67 staining of MGC80-3 or HGC-27 xenografts. Scale bar = 100 μm.


Figure 3 ( B ) lncPSCA inhibits invasion abilities of MGC80 - 3 and HGC - 27 cells . Cells on the lower surface of the chamber were stained by crystal violet . Scale bar = 100 μm . ( C ) In MGC80 - 3 and HGC - 27 cells , expression changes of different markers ( β - catenin , ZO - 1 , SNAIL and VIMENTIN ) of epithelial - to - mesenchymal transition were examined after overexpression or knockout of lncPSCA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm.  (C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA.


Figure 4 ( F ) Gastric cancer cells after lncPSCA - knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide ( CHX ) or vehicle for the indicated periods of time . DDX5 levels were analyzed by Western blot . ( I ) Knockdown of DDX5 with siRNA ( siD - 1 and siD - 2 ) substantially reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . ( J ) Silencing of DDX5 with shRNA ( shD ) significantly reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot.  (I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (J) Silencing of DDX5 with shRNA (shD) significantly reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (


Figure 5 ( E ) RNA Pol II ChIPseq and RNAseq signals of PMAIP1 , CDKN1A , SESN2 and SESN3 in HGC - 27 cells . Red bars represent the RNA Pol II ChIPseq signal . Orange or blue bars represent the RNAseq signals of HGC - 27 cells overexpressing lncPSCA ( lncP ) or with silenced DDX5 ( shD ) , respectively . ( F ) Validation of candidate downstream genes of the lncPSCA - DDX5 axis identified through the integrated analyses . qRT - PCR was performed for PMAIP1 , CDKN1A and SESN2 genes upon overexpression of lncPSCA ( lncP ) , silencing of DDX5 ( shD ) or lncPSCA konckout . Error bars indicate the SEM . Data show one representative example of three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 (E) RNA Pol II ChIPseq and RNAseq signals of PMAIP1, CDKN1A, SESN2 and SESN3 in HGC-27 cells. Red bars represent the RNA Pol II ChIPseq signal. Orange or blue bars represent the RNAseq signals of HGC-27 cells overexpressing lncPSCA (lncP) or with silenced DDX5 (shD), respectively.  (F) Validation of candidate downstream genes of the lncPSCA-DDX5 axis identified through the integrated analyses. qRT-PCR was performed for PMAIP1, CDKN1A and SESN2 genes upon overexpression of lncPSCA (lncP), silencing of DDX5 (shD) or lncPSCA konckout. Error bars indicate the SEM. Data show one representative example of three biological replicates.


Fig 1 ( A ) Swelling of neurons in the vestibular nucleus due to fine vesiculation ( arrows ) and clear vacuolization ( arrowhead ) of the cytoplasm . HE , scale bar 100 μm . ( B ) Clear cytoplasmic vacuolization ( arrows ) in cerebellar corticalPurkinje cells . HE , scale bar 100 μm . ( C ) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells ( arrows ) and a dense granular cell layer . HE , scale bar 100 μm . ( D ) Marked neuronal loss is present the cerebellar cortex of an affected dog . The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain ( arrow ) . HE , scale bar 100 μm . ( E ) Axonal spheroids of varying quality were seen in the white matter ( arrows ) of cerebellum and brainstem . HE , scale bar 100 μm . ( F ) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells . HE , scale bar 100 μm . ( G ) Purkinje cell with numerous single - membrane bound , cytoplasmic vacuoles tethering to each other ( arrows , inset ) . Electronmicrograph , scale bar 2 μm . Inset : scale bar 1 μm . ( H ) Axonal spheroid containing aggregated degenerated mitochondria , occasional double - membrane - bound autophagosomes ( arrows ) and free electron dense material , compressed by a peripheral clear vacuolar space . Electronmicrograph , scale bar 0 . 5 μm . Abbreviations : n , nucleus ; ml , molecular layer ; pl , Purkinje cell layer ; gl , granular cell layer .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 1(A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm.(B) Clear cytoplasmic vacuolization (arrows) in cerebellar corticalPurkinje cells. HE, scale bar 100 μm.(C) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm.(E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm.(F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm.(G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm.(H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer.


Fig 4 ( A ) Axonal spheroids stain diffusely positive for LC3B . IHCLC3B , scale bar 100 μm . ( B , C ) The granular cores of the spheroids are positive for ( B ) ubiquitin ( arrow ) and ( C ) p62 . IHCubiquitin and p62 , scale bars 20 and 100 μm , respectively . ( D ) Smooth axonal swellings in the cerebellar cortex contain ATG4D . Inset : control . IHCATG4D , scale bar 100 μm . ( E ) Affected neurons show increased perinuclear granular LC3B positivity . Inset : control . IHCLC3B , scale bar 100 μm . ( F ) Neuronalvacuoles are partially LAMP2 positive ( arrow ) and partially negative ( arrow head ) . Inset : control . IHCLAMP2 , scale bar 20 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 4(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.


Fig 5 ( A - F ) Lateral views of control and morphant zebrafishembryos . ( A , B ) Control embryos appear normal at 1 dpf . ( C , D ) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf . Black arrowheads denote the developing cerebellum . Purple arrows indicate the hindbrain irregularities . ( E ) Control embryo at 2 dpf . ( F ) A morphant embryo at 2 dpf displaying hydrocephalus ( yellow arrow ) and small head and eye . ( G ) RT - PCR assay showing the efficiency of the atg4daSMO .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O

Fig 5(A-F) Lateral views of control and morphant zebrafishembryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye.(G) RT-PCR assay showing the efficiency of the atg4daSMO.


Fig 6Immunostaining with ( A - C ) anti - Pvalb7 and ( D - F ) anti - zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants . ( A , D ) Control embryos at 4 . 5 dpf . ( B , E ) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells . ( C , F ) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons , which are laterally located in the cerebellum . ( G - I ) Labeling of cerebellar granule cells with anti - Vglut1 antibody in control and morphant embryos . ( H ) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum . ( I ) Vglut1 expression is strongly reduced in embryos showing severe phenotype . White arrowheads indicate the region of cerebellum . Abbreviations : hb , hindbrain .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 6Immunostaining with (A-C) anti-Pvalb7 and (D-F) anti-zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants. (A,D) Control embryos at 4.5 dpf. (B,E) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells. (C,F) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons, which are laterally located in the cerebellum. (G-I) Labeling of cerebellar granule cells with anti-Vglut1 antibody in control and morphant embryos. (H) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum. (I) Vglut1 expression is strongly reduced in embryos showing severe phenotype. White arrowheads indicate the region of cerebellum. Abbreviations: hb, hindbrain.


Fig 1A ) Transmission electron microscopy ( TEM ) of MoDCs infected with P . gingivalis for 2 , 12 and 24 hours ( left , middle and right panels ) . The sections show the intra - and extra - cellular contents of Cont . ( un - infected ) , Pg381 , Mfa1 + Pg and FimA + Pg infected MoDCs for the different time pointsB ) The figure shows the intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re - suspended and maintained in anaerobic broth for 5 days . The data represents CFU within MoDCs harvested from three healthy individuals . The means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups and Tukey ' s test for multiple group comparisons within 3 different experiments ( * statistical significance at the p < 0 . 001 ) . B ) The figure shows the intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re - suspended and maintained in anaerobic broth for 5 days . The data represents CFU within MoDCs harvested from three healthy individuals . The means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups and Tukey ' s test for multiple group comparisons within 3 different experiments ( * statistical significance at the p < 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 1A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time pointsB) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).


Fig 2A ) DC - SIGN mRNA expression in P . gingivalis - infected MoDCs at 0 . 1 , 1 and 10 MOIs . The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections . The target gene ( DC - SIGN ) was normalized using the endogenous control GAPDH ( ΔCt ) and fold regulations were calculated using 2 - ( ΔΔCt ) method . The statistical analysis was performed using the t - test , which accounts for the clustering of infected and un - infected controls within 3 different experiments ( * p < 0 . 001 ) . B ) Immuno - electron microscopy of un - infected MoDCs ( Cont . ) ( upper panel ) , MoDCs infected with Pg381 ( middle panel ) and Mfa1 + Pg mutants ( lower panel ) . Gold particles ( marked with red rings ) for positive DC - SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1 + Pg strains . Minimal positive staining for DC - SIGN was detected in the membranes of MoDCs infected with Pg381 , while no cytoplasmic gold labeling was detected in these cells . C ) Flow cytometry analysis of surface DC - SIGN in human MoDCs after infection with Pg381 , Mfa1 + Pg and FimA + Pg . The analysis of the intensity used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons 3 different experiments ( * p < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 2A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001).B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).


Fig 3A ) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with / without Rapamycin treatment . The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days . The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals ( * P < 0 . 001 ) . The analysis of readings used One - way AVOVA analysis of different groups and Tukey ' s test for multiple comparisons . B ) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P . gingivalis infections . LC3 - II ( red - fluorescent dye ) and the bacterial strains ( green CFSE ) were studied in MoDCs 11 hours after Rapamycin treatment ( 12 hours after infections ) . C ) Quantifications of the fluorescent intensity of CFSE - labeled P . gingivalis and LC3 - II signals within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons ( * P < 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 3A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons (* P<0.001).


Fig 4A ) Survival of P . gingivalis strains within MoDCs after 6 , 12 , 24 and 48 hours . Blue lines show P . gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines . The effect of rapamycin on Mfa1 + Pg , Pg381 and FimA + Pg survival within MoDCs are shown in figures B , C and D respectively . A three - factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading . The survival curves for the strains are showed in blue , while the effect of rapamycin treatments are in red . Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black , respectively . Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain ( Mfa1 + Pg , Pg381 and FimA + Pg ) between treated ( rapamycin ) and untreated were significantly different overtime ( p - value < 0 . = 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 4A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).


Fig 5A ) Epifluorescence microscopy images of MoDCs infected with Pg381 , Mfa1 + Pg , FimA + Pg and MFB strains after 12 hours . LC3 - II was detected in red - fluorescent ( Texas red ) dye and the bacterial strains were pre - labeled with CFSE ( green ) . Co - localization of P . gingivalis and LC3 - II showed in the right panels . B ) Quantifications of the fluorescent intensity of LC3 - II within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons of three different experiments ( * p < 0 . 001 , # p < 0 . 01 ) . C ) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity ( * p < 0 . 001 , # p < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 5A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels.B) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons of three different experiments (* p<0.001, # p<0.01).C) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).