Figure 4B . Percentage of transplanted ( female ) cells in the blood . n = 18 - 19 mice / group . C . Percentage of transduced ( GFP + ) cells in the blood . Mean ± SEM . * * * p < 0 . 0001 . n = 22 - 25 mice / group . D . Percentages of GFP + cells labeled with CD11b , CD19 , and CD3 . The proportion of GFP + cells labeled with CD11b is increased in Sema3F mice ( p = 0 . 01 ) , while that of CD19 - labeled cells is decreased ( p = 0 . 0003 ) . Mean ± SEM . Unpaired Student t - test . n = 9 mice / group . * p < 0 . 05 , * * * p < 0 . 001 . E . Percentages of total blood cells expressing CD11b , CD19 , and CD3 . Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice . n = 9 mice / group . Mean ± SEM .

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 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 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

Figure 4B. Percentage of transplanted (female) cells in the blood. n=18-19 mice/group.C. Percentage of transduced (GFP+) cells in the blood . Mean±SEM. ***p<0.0001. n=22-25 mice/group.D. Percentages of GFP+ cells labeled with CD11b, CD19, and CD3. The proportion of GFP+ cells labeled with CD11b is increased in Sema3F mice (p=0.01), while that of CD19-labeled cells is decreased (p=0.0003). Mean±SEM. Unpaired Student t-test. n=9 mice/group. *p<0.05,***p<0.001.E. Percentages of total blood cells expressing CD11b, CD19, and CD3. Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice. n=9 mice/group. Mean±SEM.


Figure 5A - F . Co - labeling for GFP and MBP on spinal cord sections of GFP ( A - C ) and Sema3F - GFP chimeras ( D - F ) sacrificed at 7 ( A , D ) , 10 ( B , E ) , and 60 ( C , F ) days post lesion ( dpl ) . Absence of MBP labeling in A - B , D - E indicates the lesion ( delimited by yellow lines ) . Areas faintly labeled with MBP in C - F likely represent remyelinated areas . G . Quantification of GFP + cells . Kruskal - Wallis test p = 0 . 02 . No significant differences between GFP and Sema3F at any of the time points ( Dunn ' s post test ; Mann - Whitney test ) . Decrease in GFP + cells between 7 and 60 dpl for both GFP and Sema3F mice 2m . H - M . Co - labeling for GFP / MBP / Iba1 ( macrophage / microglia marker ) shows colocalization of GFP with Iba1 . Arrows indicate double - labeled cells . N - P Co - labeling for GFP and T cell marker CD3 . Arrows indicate CD3 - labeled cells . Q - S . Co - labeling for GFP and B cell marker CD45R / B220 . Arrows indicate CD45R / B220 - labeled cells . T . Percentages of GFP + cells that are Iba1 + , CD3 + , and CD45R / B220 + . The absolute majority of GFP + cells are Iba1 + . U . Quantification of Iba1 + , CD3 + , and B220 + cells . For all 3 populations , no significant differences in cell numbers were observed between GFP and Sema3F mice , and a general decrease was observed at 60 dpl compared to earlier time points . V - Aa . Co - labeling for GFP and microglial marker P2Y12 . Purple arrows indicate P2Y12 + cells . Exclusion between the two markers is obvious in both groups . Ab . Quantification of P2Y12 + cells . No significant changes are observed between the groups or across the time .

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 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 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 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 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 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

Figure 5A-F. Co-labeling for GFP and MBP on spinal cord sections of GFP (A-C) and Sema3F-GFP chimeras (D-F) sacrificed at 7 (A,D), 10 (B,E), and 60 (C,F) days post lesion (dpl). Absence of MBP labeling in A-B,D-E indicates the lesion (delimited by yellow lines). Areas faintly labeled with MBP in C-F likely represent remyelinated areas.G. Quantification of GFP+ cells. Kruskal-Wallis test p=0.02. No significant differences between GFP and Sema3F at any of the time points (Dunn's post test; Mann-Whitney test). Decrease in GFP+ cells between 7 and 60 dpl for both GFP and Sema3F mice 2m.H-M. Co-labeling for GFP/MBP/Iba1 (macrophage/microglia marker) shows colocalization of GFP with Iba1. Arrows indicate double-labeled cells.N-P Co-labeling for GFP and T cell marker CD3. Arrows indicate CD3-labeled cells.Q-S. Co-labeling for GFP and B cell marker CD45R/B220. Arrows indicate CD45R/B220-labeled cells.T. Percentages of GFP+ cells that are Iba1+, CD3+, and CD45R/B220+. The absolute majority of GFP+ cells are Iba1+.U. Quantification of Iba1+, CD3+, and B220+ cells. For all 3 populations, no significant differences in cell numbers were observed between GFP and Sema3F mice, and a general decrease was observed at 60 dpl compared to earlier time points.V-Aa. Co-labeling for GFP and microglial marker P2Y12. Purple arrows indicate P2Y12+ cells. Exclusion between the two markers is obvious in both groups. Ab. Quantification of P2Y12+ cells. No significant changes are observed between the groups or across the time.


Figure 6B - G . Lesions at 7 dpl in GFP ( B , E ) and Sema3F ( C , F ) mice . B - C . Co - labeling for PDGFRα , MBP , and DAPI . Arrows indicate OPCs in the lesion . Bi and Ci - insets of white dotted squares in B and C . D . Higher numbers of PDGFRα + ( OPCs ) cells in Sema3F mice at 7 dpl ( Mann Whitney test : p = 0 . 0031 , n = 6 - 8 mice / group ) . E - F . Co - labeling for Olig2 and MBP . E ' - F ' . Co - labeling for Olig2 and GFP of the lesions shown in E - F . 3D reconstructions . Proximity of OPCs and Sema 3F - carrying cells . E ' i - E ' iii and F ' i - F ' iii - insets of E ' - F ' . G . Higher numbers of Olig2 + ( oligodendroglial ) cells in Sema3F - GFP mice at 7 dpl ( Mann Whitney test : p = 0 . 0047 , n = 5 - 8 mice / group ) . H - I . Immunolabelling for activated OPC marker Nkx2 . 2 . J . Numbers of Nkx2 . 2 + cells are increased in lesions of Sema3F - GFP mice at 7dpl ( Mann Whitney test : p = 0 . 014 , n = 6 - 7 mice / group ) . Arrows indicate Nkx2 . 2 + cells in close proximity of GFP + cells . K - P . colabelling for Nkx2 . 2 and proliferation marker Ki67 . Arrows indicate double labeled cells . Q . Numbers of Nkx2 . 2 + Ki67 + cells are increased at 7 dpl in Sema3F - GFP mice . Mean ± SEM . n = 6 - 7 mice / group . R . The proportion of Nkx2 . 2 + cells co - expressing Ki67 is unchanged . Mean ± SEM . n = 6 - 7 mice / group . S - X . Colabelling for Olig2 and cleaved caspase 3 , marker of apoptosis indicates occasional apoptotic cells in both groups of mice that are Olig2 - . Arrows indicate cleaved caspase 3 + cells . Y . Numbers of cleaved caspase 3 + cells are the same in two groups . Mean ± SEM . n = 6 mice / group .

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 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 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 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

Figure 6B-G. Lesions at 7 dpl in GFP (B,E) and Sema3F (C,F) mice. B-C. Co-labeling for PDGFRα, MBP, and DAPI. Arrows indicate OPCs in the lesion. Bi and Ci-insets of white dotted squares in B and C. D. Higher numbers of PDGFRα+ (OPCs) cells in Sema3F mice at 7 dpl (Mann Whitney test: p=0.0031, n=6-8 mice/group). E-F. Co-labeling for Olig2 and MBP. E'-F'. Co-labeling for Olig2 and GFP of the lesions shown in E-F. 3D reconstructions. Proximity of OPCs and Sema 3F-carrying cells. E'i-E'iii and F'i-F'iii-insets of E'-F'. G. Higher numbers of Olig2+ (oligodendroglial) cells in Sema3F-GFP mice at 7 dpl (Mann Whitney test: p=0.0047, n=5-8 mice/group).H-I. Immunolabelling for activated OPC marker Nkx2.2. J. Numbers of Nkx2.2+ cells are increased in lesions of Sema3F-GFP mice at 7dpl (Mann Whitney test: p=0.014, n=6-7 mice/group). Arrows indicate Nkx2.2+ cells in close proximity of GFP+ cells.K-P. colabelling for Nkx2.2 and proliferation marker Ki67. Arrows indicate double labeled cells. Q. Numbers of Nkx2.2+Ki67+ cells are increased at 7 dpl in Sema3F-GFP mice. Mean±SEM. n=6-7 mice/group. R. The proportion of Nkx2.2+ cells co-expressing Ki67 is unchanged. Mean±SEM. n=6-7 mice/group.S-X. Colabelling for Olig2 and cleaved caspase 3, marker of apoptosis indicates occasional apoptotic cells in both groups of mice that are Olig2-. Arrows indicate cleaved caspase 3+ cells.Y. Numbers of cleaved caspase 3+ cells are the same in two groups. Mean±SEM. n=6 mice/group.


Figure 7A - B . Labelling for APC / CC1 , a marker of oligodendrocytes . Dotted lines indicate the lesion border . APC + cells are scarce in the lesions of GFP mice ( A ) . B . APC + cells in the lesion of Sema3F mice . C . Quantification of APC + cells ( Mann Whitney test ; p = 0 . 0061 for 7 dpl , p = 0 . 0081 for 10 dpl ; n = 6 - 7 mice / group ) . D - E . EM images of the lesion in GFP ( D ) and Sema3F ( E ) chimeras at 14 dpl . Thin myelin sheaths in Sema 3F mice ( yellow arrows ) . F . Higher power image of a new myelin sheath in a Sema3F mouse . Fi . Inset of yellow square in F . G . Quantification of remyelinated axons ( Mann Whitney test : p = 0 . 03 , n = 6 mice / group ) . * p < 0 . 05 . H . In Sema3F mice , oligodendroglial cells ( O ) and early remyelination ( * ) are frequently observed in macrophage ( M ) vicinity . Hi . Inset of yellow dashed square in H .

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 O O O O O O O O O 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 7A-B. Labelling for APC/CC1, a marker of oligodendrocytes. Dotted lines indicate the lesion border. APC+ cells are scarce in the lesions of GFP mice (A). B. APC+ cells in the lesion of Sema3F mice. C. Quantification of APC+ cells (Mann Whitney test; p=0.0061 for 7 dpl, p=0.0081 for 10 dpl; n= 6-7 mice/group).D-E. EM images of the lesion in GFP (D) and Sema3F (E) chimeras at 14 dpl. Thin myelin sheaths in Sema 3F mice (yellow arrows). F. Higher power image of a new myelin sheath in a Sema3F mouse. Fi. Inset of yellow square in F. G. Quantification of remyelinated axons (Mann Whitney test: p=0.03, n=6 mice/group). *p<0.05. H. In Sema3F mice, oligodendroglial cells (O) and early remyelination (*) are frequently observed in macrophage (M) vicinity. Hi. Inset of yellow dashed square in H.


Figure 1 ( B ) Representative images of immunostaining demonstrate that FKRP - iPSCs express specific pluripotency - associated markers , including NANOG , OCT4 , Tra - 1 - 60 and SSEA4 . Data information : Scale bars , 50 μm . ( C ) FKRP - iPSCs have a normal karyotype . Data information : Scale bars , 50 μm . ( D ) In vitro differentiation of FKRP - iPSCs to cells representing ectoderm ( β - III Tubulin , Tuj1 ) , mesoderm ( SMA , smooth muscle actin ) and endoderm ( AFP , α - fetoprotein ) . Data information : Scale bars , 50 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 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 O O O O O O O O O O O O

Figure 1(B) Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60 and SSEA4. Data information: Scale bars, 50 μm.(C) FKRP-iPSCs have a normal karyotype. Data information: Scale bars, 50 μm.(D) In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (β-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin) and endoderm (AFP, α-fetoprotein). Data information: Scale bars, 50 μm.


Figure 2 ( C ) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones ( 5D17 , 5D23 and 3B17 ) , compared with their parental FKRPA455D - iPSCs . Selection cassette excision sites are identified in the corrected - iPSC lines . ( D ) CRISPR / Cas9 corrected - iPSC lines show a normal karyotype .

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

Figure 2(C) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones (5D17, 5D23 and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines.(D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.


Figure 3 ( A and B ) Representative images of NSCs derived from FKRP - and corrected - iPSC lines expressing SOX1 , SOX2 and nestin . ( C and D ) Quantification of percentage of SOX1 + ( C ) and SOX2 + ( D ) cells in culture . The efficiency of neural induction is more than 99 % in FKRP - and corrected - iPSC ( 5D17 , 5D23 and 3B17 ) lines . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm . ( E and F ) FKRP - and corrected NSC lines can be further differentiated to cortical neural progenitor cells , expressing PAX6 , OTX2 and vimentin . ( G - I ) Quantification of percentage of PAX6 + ( G ) and OTX2 + ( H ) cells in culture . About 91 - 98 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express PAX6 ( G ) . About 93 - 96 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express OTX2 ( H ) . Of the OTX2 + population , about 60 - 67 % cells are also Ki67 + cycling progenitors ( I ) . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm . ( J and K ) Glutamatergic projection neurons derived from FKRP and corrected ( 5D17 , 5D23 and 3B17 ) progenitor cells . The vast majority of neurons contain vGlut1 + punctae in their neurites ( labelled by Tuj1 ) . Right panels are enlarged images from the insets of left panels . Data information : Scale bars , 50 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 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 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

Figure 3(A and B) Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2 and nestin. (C and D) Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23 and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.(E and F) FKRP- and corrected NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2 and vimentin. (G-I) Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.


Figure 4 ( A ) Molecular weight of glycosylated α - dystroglycan ( IIH6 epitope ) in WT - iPSC derived cortical neurons is similar to that in the mouse brain ( ~ 120 kDa ) and lower than that in the mouse muscle ( ~ 156kDa ) . IIH6 reactivity was almost not detected in cortical neurons derived from FKRP - iPSCs . Note that the β - Actin antibody does not cross - react with the muscle sample . ( B ) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC - derived cortical neurons . Intensities of IIH6 reactivity are normalized to β - Actin expression , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * * p < 0 . 01 ) ( C ) Representative laminin overlays show that targeted gene correction restores laminin - binding activity in iPSC - derived cortical neurons . Intensities of laminin - binding activity are normalized to β - dystroglycan , indicating mean ± s . d . ( n = 3 biological replicates ; 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 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 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

Figure 4(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.(B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).


Figure 5 ( C ) Dose response curve . H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed , stained and imaged as described above . The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non - linear regression curve was fitted for the 3 compounds showing the highest increase in α - dystroglycan glycosylation ( IIH6 immunostaining ) . Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown . HPFPTzone : ( 5Z ) - 5 - [ ( 3 - ethoxy - 4 - hydroxy - phenyl ) methylene ] - 3 - ( 4 - fluorophenyl ) - 2 - thioxo - thiazolidin - 4 - one . 4BPPNit : 4 - ( 4 - bromophenyl ) - 6 - ethylsulfanyl - 2 - oxo - 3 , 4 - dihydro - 1H - pyridine - 5 - carbonitrile . DPPTzone : ( 5E ) - 5 - [ ( 2 , 5 - dimethyl - 1 - phenyl - pyrrol - 3 - yl ) methylene ] - 3 - phenyl - 2 - thioxo - thiazolidin - 4 - one . Regression curve and EC50 values were calculated using GraphPad Prism 7 software . In images shown , green represents glycosylated α - dystroglycan and blue Hoechst staining . Scale bar , 50 µm . ( E ) Flow cytometry analysis confirmed that 4BPPNit treatment ( 20 µM ) significantly increased IIH6 - reactive glycan in mouse H2K 2B4 cells , compared with untreated cells . There was no significant difference in the percentage of IIH6 - positive cells . Values indicate mean ± s . e . m . ( n = 6 , triplicates of 2 independent experiments ; t - test ; NS , not significant ; * * p < 0 . 01 ) .

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 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

Figure 5(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.(E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).


Figure 6 ( A ) FKRP - NSCs treated with 4BPPNit showed increased IIH6 reactivity , compared with untreated control cells . Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR - corrected NSCs . Under the same exposure time , the signal intensity of IIH6 reactivity is saturated in corrected - NSCs . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . ( B ) Representative immunoblots with the IIH6 antibody using FKRP - and corrected - NSCs treated with 4BPPNit for 24 , 48 and 72 h , and DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time . ( C ) Quantification of α - dystroglycan glycosylation ( IIH6 ) , compared with DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) . ( D ) Representative immunoblots of laminin - binding analysis using FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( E ) Quantification of laminin - binding activities , compared with DMSO only controls . Data information : Intensities of laminin - binding activity are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) . ( F ) Representative immunoblots with the AF6868 antibody detecting both α - dystroglycan core protein and β - dystroglycan in FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( G ) Quantification of α - dystroglycan core protein and β - dystroglycan expression , compared with DMSO only controls . Data information : Intensities of protein expression are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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 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 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 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

Figure 6(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression.(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time.(C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).(D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


Figure 7 ( A ) DAG1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ( B ) FKRP gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) . ( C ) LARGE1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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 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 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 7(A) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01(B) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).(C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


Figure 1B - D . Mice were analyzed by Kaplan - Meier survival plot ( B ) , the percentage of body weight ( C ) , and the clinical scores ( D ) of WT and GPx8 - / - mice with colitis induced by 4 % DSS for 6 days ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( E ) Images and statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . , 5 - 6 mice were used for each experiment . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections . The infiltrated immune cells at the transmural extensions were indicated by arrowhead . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( G ) Colon sections were stained with markers for macrophages ( F4 / 80 ) and anti - GPx8 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing ( n = 5 per group ) and are presented by a heat map , ( I ) , and principal coordinates analysis , ( J ) . Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . ( K ) α - diversity indicated bacteria species in each mouse depicted by richness ( total operational taxonomic units , OTUs ) , the Chao1 richness estimator ( Chao1 ) , and the abundance - based coverage estimator ( ACE ) metrics . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Figure 1B-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. , 5-6 mice were used for each experiment. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing (n = 5 per group) and are presented by a heat map, (I), and principal coordinates analysis, (J).Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. (K) α-diversity indicated bacteria species in each mouse depicted by richness (total operational taxonomic units, OTUs), the Chao1 richness estimator (Chao1), and the abundance-based coverage estimator (ACE) metrics. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Figure 2B . Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages ( F4 / 80 ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . ( C ) The percentage of body weight . ( D ) The clinical scores of mice . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( E ) Statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( G ) Colon sections stained with anti - F4 / 80 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . D ) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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 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 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 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 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 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

Figure 2B. Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages (F4/80).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). (C) The percentage of body weight. (D) The clinical scores of mice. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (E) Statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (F) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (G) Colon sections stained with anti-F4/80. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). D) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


Figure 3A . Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency . BMDMs were primed for 6 h with 0 . 5 μg / ml LPS and then transfected with LPS ( TF LPS ) for 8 h , stimulated with ATP ( 5 mM ) for 3 h , MSU ( 150 μg / mL ) for 8 h , or nigericin ( 5 μM ) for 3 h . Activation of inflammasome was demonstrated by secreted IL - 1β . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . B . GPx8 deficiency enhanced noncanonical inflammasome activation . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . C . Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs . BMDMs were primed for 6 h with 1 μg / ml poly ( I : C ) and then transfected with LPS for 8 h . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( D ) Activation of noncanonical inflammasome in BMDMs with empty vector ( EV ) or hGPx8 was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( E ) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . F . GPx8 deficiency enhanced caspase - 11 cleavage and activation . Levels of processed caspase - 1 and - 11 in the supernatants ( Sup ) and full - length caspase - 1 , - 11 and GAPDH in the cell lysates ( Cell ext ) were determined by immunoblotting . Fold changes of protein expression were normalized with internal controls and are indicated below the blots . G . RNA expression of caspase - 11 was not altered by GPx8 deficiency . Caspase - 11 RNA expression of BMDMs isolated from GPx8 - / - mice was measured after LPS priming . Data information : ata are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . H . GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo . Colon samples from 5 WT or GPx8 - / - mice treated with DSS for 5 days were collected on day 7 . Levels of full - length and processed caspase - 1 , - 11 and gasdermin D ( GSDMD ) in tissue lysates were individually analyzed by immunoblots . GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( I ) Survival of mice primed with ultra pure LPS ( E . coli O55 : B5 , 400 μg / kg ) and re - challenged 6 h later with LPS ( 40 mg / kg ) ( n = 8 per group ) . GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( J ) Survival of mice primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 10 mg / kg ) ( n = 9 per group ) . GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( K ) IL - 1β production levels in mice sera were measured 2 h after being primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 1 mg / kg ) ( n = 8 - 9 per group ) . In ( K ) , data are presented as the mean ± SEM . NS , no significance . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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 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 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 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 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 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 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 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 O 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

Figure 3A. Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency. BMDMs were primed for 6 h with 0.5 μg/ml LPS and then transfected with LPS (TF LPS) for 8 h, stimulated with ATP (5 mM) for 3 h, MSU (150 μg/mL) for 8 h, or nigericin (5 μM) for 3 h. Activation of inflammasome was demonstrated by secreted IL-1β. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).B. GPx8 deficiency enhanced noncanonical inflammasome activation. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).C. Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs. BMDMs were primed for 6 h with 1 μg/ml poly(I:C) and then transfected with LPS for 8 h. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (D) Activation of noncanonical inflammasome in BMDMs with empty vector (EV) or hGPx8 was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (E) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.F. GPx8 deficiency enhanced caspase-11 cleavage and activation. Levels of processed caspase-1 and -11 in the supernatants (Sup) and full-length caspase-1, -11 and GAPDH in the cell lysates (Cell ext) were determined by immunoblotting. Fold changes of protein expression were normalized with internal controls and are indicated below the blots.G. RNA expression of caspase-11 was not altered by GPx8 deficiency. Caspase-11 RNA expression of BMDMs isolated from GPx8-/- mice was measured after LPS priming. Data information: ata are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.H. GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo. Colon samples from 5 WT or GPx8-/- mice treated with DSS for 5 days were collected on day 7. Levels of full-length and processed caspase-1, -11 and gasdermin D (GSDMD) in tissue lysates were individually analyzed by immunoblots.GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (I) Survival of mice primed with ultra pure LPS (E. coli O55:B5, 400 μg/kg) and re-challenged 6 h later with LPS (40 mg/kg) (n = 8 per group).GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (J) Survival of mice primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (10 mg/kg) (n = 9 per group).GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (K) IL-1β production levels in mice sera were measured 2 h after being primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (1 mg/kg) (n = 8-9 per group). In (K), data are presented as the mean ± SEM. NS, no significance. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Figure 4A . Interaction of GPx8 with human caspase - 4 ( Casp4 ) or mouse caspase - 11 ( Casp11 ) was confirmed by co - IP assays . 293T cells were co - transfected with vectors expressing FLAG tagged Casp4 or Casp11 , in addition to GPx8 expressing vectors . Cell lysate containing Casp4 - or Casp11 - FLAG proteins was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 and anti - FLAG Ab ( left panel ) . GPx8 and its interacting proteins were precipitated by anti - GPx8 Ab and analyzed by the indicated Abs ( right panel ) . B . Co - localization of Myc - tagged GPx8 ( red ) and endogenous caspase - 4 ( green ) in THP - 1 cells . C . Interaction between GPx8 and caspase - 4 in THP - 1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay ( PLA ) ( red dots , labeled by arrowheads ) . GPx8 - Myc stably expressing THP - 1 were used to detect the interaction between GPx8 - Myc and endogenous caspase - 4 . Untreated cells ( Ctrl ) or cells primed with LPS ( primed LPS ) were incubated with anti - Myc and anti - caspase - 4 Abs , according to the manufacturer ' s instructions , and the nucleus was stained with DAPI ( blue ) . Results were quantified by counting at least 5 different fields with an average of 300 cells . Reactions without primary Abs were used as the control for the PLA assay ( PLA Ctrl ) . Data information : All data are representative of at least 3 independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . 293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( D ) Complexes of GPx8 and caspase - 4 were demonstrated by co - IP assays . Cysteine - dependent covalent interactions of GPx8 and caspase - 4 ( C258S ) were analyzed under non - reducing and reducing conditions . Disulfide - linked complexes of Gpx8 and caspase - 4 are indicated . 293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( E ) Interactions of GPx8 and caspase - 4 in response to H2O2 treatment were demonstrated by co - IP assays of lysates prepared from 293T cells treated with H2O2 ( 200 μM ) . 293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( F ) C79S , C108S , and C2S2 mutants of GPx8 abolished most of the binding to caspase - 4 by co - IP assays . C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . THP - 1 cells stably expressing empty vector ( EV ) , WT , or mutant GPx8 proteins were confirmed by immunoblotting ( G ) . C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . ( H and I ) Cells were treated with 12 - O - tetradecanoylphorbol 13 - actate ( PMA ) ( 0 . 1 μM ) and differentiated for 3 days then primed and transfected with LPS . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( H ) and LDH release ( I ) . Data information : All data are representative of at least 3 independent experiments . In H and I , data are presented as the mean ± SD , n = 4 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . J . E . coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase - 4 ( C258S ) to form a disulfide - linked complex ( oxidized GPx8 : oGPx8 ; reduced GPx8 : rGPx8 ) . Lanes 2 and 3 , GPx8 pretreated with 1 and 5 mM H2O2 , respectively . Lanes 4 and 5 , Gpx8 pretreated with 50 and 200 mM DTT , respectively . Disulfide - linked complexes of recombinant Gpx8 and caspase - 4 ( C258S ) are indicated . K . GPx8 inhibited the activation of caspase - 4 by decreasing the oligomerization of caspase - 4 . Oxidized GPx8 and caspase - 4 ( C258S ) were co - incubated for 30 min . Different amounts of LPS were added to the complex and incubated for another 30 min . Oligomerized caspase - 4 was analyzed by native blue PAGE according to the manufacturer ' s instructions . Mock : without GPx8 ; oxidized GPx8 : oGPx8 ; oxidized GPx8C79S : oC79S . Data information : All data are representative of at least 3 independent experiments . In K , quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers / total proteins . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A. Interaction of GPx8 with human caspase-4 (Casp4) or mouse caspase-11 (Casp11) was confirmed by co-IP assays. 293T cells were co-transfected with vectors expressing FLAG tagged Casp4 or Casp11, in addition to GPx8 expressing vectors. Cell lysate containing Casp4- or Casp11-FLAG proteins was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 and anti-FLAG Ab (left panel). GPx8 and its interacting proteins were precipitated by anti-GPx8 Ab and analyzed by the indicated Abs (right panel).B. Co-localization of Myc-tagged GPx8 (red) and endogenous caspase-4 (green) in THP-1 cells.C. Interaction between GPx8 and caspase-4 in THP-1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay (PLA) (red dots, labeled by arrowheads). GPx8-Myc stably expressing THP-1 were used to detect the interaction between GPx8-Myc and endogenous caspase-4. Untreated cells (Ctrl) or cells primed with LPS (primed LPS) were incubated with anti-Myc and anti-caspase-4 Abs, according to the manufacturer's instructions, and the nucleus was stained with DAPI (blue). Results were quantified by counting at least 5 different fields with an average of 300 cells. Reactions without primary Abs were used as the control for the PLA assay (PLA Ctrl). Data information: All data are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (D) Complexes of GPx8 and caspase-4 were demonstrated by co-IP assays. Cysteine-dependent covalent interactions of GPx8 and caspase-4 (C258S) were analyzed under non-reducing and reducing conditions. Disulfide-linked complexes of Gpx8 and caspase-4 are indicated.293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (E) Interactions of GPx8 and caspase-4 in response to H2O2 treatment were demonstrated by co-IP assays of lysates prepared from 293T cells treated with H2O2 (200 μM).293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (F) C79S, C108S, and C2S2 mutants of GPx8 abolished most of the binding to caspase-4 by co-IP assays.C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. THP-1 cells stably expressing empty vector (EV), WT, or mutant GPx8 proteins were confirmed by immunoblotting (G).C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. (H and I) Cells were treated with 12-O-tetradecanoylphorbol 13-actate (PMA) (0.1 μM) and differentiated for 3 days then primed and transfected with LPS. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (H) and LDH release (I). Data information: All data are representative of at least 3 independent experiments. In H and I, data are presented as the mean ± SD, n=4, technical repeats. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).J. E. coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase-4 (C258S) to form a disulfide-linked complex (oxidized GPx8: oGPx8; reduced GPx8: rGPx8). Lanes 2 and 3, GPx8 pretreated with 1 and 5 mM H2O2, respectively. Lanes 4 and 5, Gpx8 pretreated with 50 and 200 mM DTT, respectively. Disulfide-linked complexes of recombinant Gpx8 and caspase-4 (C258S) are indicated.K. GPx8 inhibited the activation of caspase-4 by decreasing the oligomerization of caspase-4. Oxidized GPx8 and caspase-4 (C258S) were co-incubated for 30 min. Different amounts of LPS were added to the complex and incubated for another 30 min. Oligomerized caspase-4 was analyzed by native blue PAGE according to the manufacturer's instructions. Mock: without GPx8; oxidized GPx8: oGPx8; oxidized GPx8C79S: oC79S. Data information: All data are representative of at least 3 independent experiments. In K, quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers/total proteins. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


Figure 5A . Casp4C118S mutant partially abolished the covalent disulfide - link with GPx8 . Interacting GPx8 were co - immunoprecipitated with FLAG - tagged caspase - 4 ( WT , C118S , C329S mutants ) and analyzed under reducing ( + DTT ) or non - reducing ( - DTT ) conditions . Disulfide - linked complexes of GPx8 and caspase - 4 are indicated . Fold change of complexes was normalized with internal controls and indicated below blots . C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . THP - 1 cells stably expressing empty vector ( EV ) , FLAG - tagged caspase - 4 ( WT , C258S , C118S mutants ) , or GPx8 ( WT and C79S mutants ) were confirmed by immunoblotting ( B ) . Data information : All data shown are representative of at least 3 independent experiments . C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . ( C - D ) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS . THP - 1 cells were differentiated , primed , and transfected with LPS . Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL - 1β ( C ) and LDH ( D ) . In C and D , data are presented as the mean ± SD , n = 4 , technical repeats . * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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 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

Figure 5A. Casp4C118S mutant partially abolished the covalent disulfide-link with GPx8. Interacting GPx8 were co-immunoprecipitated with FLAG-tagged caspase-4 (WT, C118S, C329S mutants) and analyzed under reducing (+DTT) or non-reducing (-DTT) conditions. Disulfide-linked complexes of GPx8 and caspase-4 are indicated. Fold change of complexes was normalized with internal controls and indicated below blots.C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. THP-1 cells stably expressing empty vector (EV), FLAG-tagged caspase-4 (WT, C258S, C118S mutants), or GPx8 (WT and C79S mutants) were confirmed by immunoblotting (B). Data information: All data shown are representative of at least 3 independent experiments.C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. (C-D) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS. THP-1 cells were differentiated, primed, and transfected with LPS. Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL-1β (C) and LDH (D). In C and D, data are presented as the mean ± SD, n=4, technical repeats. ***P < 0.001 (Student's two‐tailed t‐test).


Figure 6A . The caspase - 4 / 11 and - 1 inhibitor , VX - 765 , inhibited noncanonical inflammasome activation . BMDMs isolated from mice were primed with LPS for 5 - 6 h , treated with 20 μM VX - 765 , and transfected with LPS for 12 h . Data information data are presented as the mean ± SD , n = 5 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and analyzed by a Kaplan - Meier survival plot ( B ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . Data information : , data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and the percentage of body weight ( C ) and clinical scores ( D ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Full - length and cleavage forms of caspase - 11 , caspase - 1 , and GSDMD ( E ) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Production of IL - 1β and IL - 6 ( F ) in colon tissue lysates was assessed on day 14 after treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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 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 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 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

Figure 6A. The caspase-4/11 and -1 inhibitor, VX-765, inhibited noncanonical inflammasome activation. BMDMs isolated from mice were primed with LPS for 5-6 h, treated with 20 μM VX-765, and transfected with LPS for 12 h. Data information data are presented as the mean ± SD, n=5, technical repeats. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and analyzed by a Kaplan-Meier survival plot (B) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). Data information: , data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and the percentage of body weight (C) and clinical scores (D) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Full-length and cleavage forms of caspase-11, caspase-1, and GSDMD (E) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Production of IL-1β and IL-6 (F) in colon tissue lysates was assessed on day 14 after treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Figure 7A . GPx8 was predominantly expressed in macrophages of UC patients . Colon biopsies of non - inflamed regions from UC patients were stained with the pan - macrophage marker , CD68 , or the monocyte / macrophage activating marker , CD163 , combined with anti - GPx8 . Quantification of double - positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double - positive cells . The total cell numbers are the sum of double - and single - positive cells . B . Relative expression levels of GPx8 , GPx7 and caspase - 4 in non - inflamed biopsy specimens from healthy controls ( Ctrl ) ( n = 14 ) and UC patients ( n = 25 ) are demonstrated by immunoblots . C . Data are normalized to the mean of healthy controls and presented as the mean ± SEM . Mann Whitney test for GPx8 and Student ' s two ‐ tailed t ‐ test for GPx7 and caspase - 4 expression analysis . Data information : NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . D . Paired GPx8 and caspase - 4 expression from each individual are linked by lines and presented as the mean ± SEM . The p - value was analyzed by the Fisher ' s exact test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * 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 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 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

Figure 7A. GPx8 was predominantly expressed in macrophages of UC patients. Colon biopsies of non-inflamed regions from UC patients were stained with the pan-macrophage marker, CD68, or the monocyte/macrophage activating marker, CD163, combined with anti-GPx8. Quantification of double-positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double-positive cells. The total cell numbers are the sum of double- and single-positive cells.B. Relative expression levels of GPx8, GPx7 and caspase-4 in non-inflamed biopsy specimens from healthy controls (Ctrl) (n = 14) and UC patients (n = 25) are demonstrated by immunoblots. C. Data are normalized to the mean of healthy controls and presented as the mean ± SEM. Mann Whitney test for GPx8 and Student's two‐tailed t‐test for GPx7 and caspase-4 expression analysis. Data information: NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.D. Paired GPx8 and caspase-4 expression from each individual are linked by lines and presented as the mean ± SEM. The p-value was analyzed by the Fisher's exact test. *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 1 . ( D ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells 56 h after transfection with the indicated siRNAs . When indicated , cells were exposed to 100 nM rapamycin for the last 3 h . RPTOR ( raptor ) and BECN1 ( beclin1 ) siRNAs served as positive and negative controls , respectively . ( E ) Autophagic flux was measured as the ratio between luciferase activities in MCF7 - RLuc - LC3WT and MCF7 - RLuc - LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h . Error bars are SDs for a representative ( n = 5 ) triplicate experiment with a minimum of 4 × 10 randomly chosen areas / sample analyzed ( D ) or three independent experiments ( F ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , as compared with similarly treated control siRNA - transfected samples .

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 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

Figure 1.(D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively.(E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.


Figure 2 . ( A ) Representative immunoblots of indicated proteins from whole - cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0 - 40 min of amino acid starvation [ − AA ] ) . ( B ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar ( siRNA - resistant CIP2A ) - encoding lentivirus , transfected with the indicated siRNAs for 53 h , and treated with 100 nM rapamycin ( Rapa ) for 0 - 20 min . ( C ) MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar - encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP - LC3 puncta formation . ( D ) MCF7 - EGFP - LC3 cells transfected with control ( − ) and CIP2A siRNAs together with an empty vector ( − ) or MYC - S62A cDNA as indicated were analyzed 56 h later for EGFP - LC3 puncta ( left ) and expression of indicated proteins ( right ) . ( E ) Representative immunoblots ( left ) of indicated proteins from whole - cell lysates of MCF7 cells treated with the control ( C ) , RPTOR ( RP ) , or CIP2A ( CIP ) siRNAs for 56 h and densitometric quantification of three independent experiments ( right ) . Error bars show SDs for three independent experiments . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , compared with Student ' s t test as indicated ( C and D ) or with control siRNA - transfected samples ( E ) . Black lines indicate that intervening lanes have been spliced out .

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

Figure 2.(A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0-40 min of amino acid starvation [−AA]).(B) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar (siRNA-resistant CIP2A)-encoding lentivirus, transfected with the indicated siRNAs for 53 h, and treated with 100 nM rapamycin (Rapa) for 0-20 min.(C) MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar-encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP-LC3 puncta formation.(D) MCF7-EGFP-LC3 cells transfected with control (−) and CIP2A siRNAs together with an empty vector (−) or MYC-S62A cDNA as indicated were analyzed 56 h later for EGFP-LC3 puncta (left) and expression of indicated proteins (right).(E) Representative immunoblots (left) of indicated proteins from whole-cell lysates of MCF7 cells treated with the control (C), RPTOR (RP), or CIP2A (CIP) siRNAs for 56 h and densitometric quantification of three independent experiments (right). Error bars show SDs for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with Student's t test as indicated (C and D) or with control siRNA-transfected samples (E). Black lines indicate that intervening lanes have been spliced out.


Figure 3 . ( A , top left ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells infected with control or CIP2A lentiviral shRNAs . Representative immunofluorescence images ( right ) and immunoblots ( bottom ) are shown . ( B ) Mean cell diameters of MCF7 - EGFP - LC3 cells infected as in A were measured with a cytometer ( NucleoCounter NC - 3000 ; Chemometec AS ) equipped with Via1 - Cassettes ( Chemometec AS ) . ( C ) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin ( Rapa ) for 72 h . The viable cells were either visualized after fixation and crystal violet staining ( left ; representative figure , n = 4 ) or trypsinized and counted with NucleoCounter NC - 3000 equipped with Via1 - Cassettes ( right ) . ( D ) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA , 2 µM MG132 , 5 µM siramesine ( Sira ) , or 100 µM etoposide ( Etop ) for 48 h , and the metabolic activity ( cell density ) was measured by the MTT assay ( left ) , and cell death was measured by the LDH assay ( right ) . ( E ) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A - encoding lentivirus . ( F ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF10A ( left ) and HEK - 293 ( right ) cells infected as in E and treated with 100 nM rapamycin for the indicated times . Black lines indicate that intervening lanes have been spliced out .

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 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 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 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, top left) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells infected with control or CIP2A lentiviral shRNAs. Representative immunofluorescence images (right) and immunoblots (bottom) are shown. (B) Mean cell diameters of MCF7-EGFP-LC3 cells infected as in A were measured with a cytometer (NucleoCounter NC-3000; Chemometec AS) equipped with Via1-Cassettes (Chemometec AS).(C) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin (Rapa) for 72 h. The viable cells were either visualized after fixation and crystal violet staining (left; representative figure, n = 4) or trypsinized and counted with NucleoCounter NC-3000 equipped with Via1-Cassettes (right).(D) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA, 2 µM MG132, 5 µM siramesine (Sira), or 100 µM etoposide (Etop) for 48 h, and the metabolic activity (cell density) was measured by the MTT assay (left), and cell death was measured by the LDH assay (right).(E) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A-encoding lentivirus.(F) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF10A (left) and HEK-293 (right) cells infected as in E and treated with 100 nM rapamycin for the indicated times. Black lines indicate that intervening lanes have been spliced out.


Figure 4 . CIP2A expression correlates with mTORC1 activity in primary human breast tumors . TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores ( arbitrary units [ a . u . ] ) for duplicate samples were averaged and plotted according to mean CIP2A expression ( left ) . The red lines are the regression lines , and the stippled red lines define the 95 % confidence intervals . Statistical analysis of data was performed using NCSS 2007 and Prism version 5 . Examples of the staining of breast cancer ( a ) and normal ( b ) cores indicated in the P - T389 - S6K1 / CIP2A plot are shown on the right . rsp , Spearman ' s correlation coefficient .

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

Figure 4.CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman's correlation coefficient.


Figure 5 . ( A and B ) In vitro translated full - length CIP2A was incubated with purified human PP2A complex and HA - Raptor ( A ) or HA - S6K1 ( B ) purified from transiently transfected MCF7 cells . Protein complexes were immunoprecipitated with anti - HA antibody and analyzed by immunoblotting as indicated . Nonspecific IgG served as a control . Note that human reticulocyte ( RC ) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac . ( C ) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right . ( D ) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control ( Ctr ) or CIP2A shRNA lentiviruses ( Fig . 3 A ) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right . ( E ) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control ( C ) or PPP2R1A ( R1A ) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies . Immunoblots of corresponding cell lysates are shown on the left . ( F ) HA - S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses ( Fig . 3 A ) , transiently transfected with HA - RPS6K1 cDNA and starved for amino acids for 20 min when indicated , were analyzed by immunoblotting . Immunoblots of corresponding cell lysates are shown on the right . ( G ) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full - length CIP2A . Error bars are SDs for four ( D ) independent experiments or a representative triplicate experiment ( C and G ) . ( H ) Representative confocal microscopy images of untreated and amino acid - starved ( 4 h ) MCF7 cells stained for endogenous CIP2A and raptor . Black lines indicate that intervening lanes have been spliced out . − AA , amino acid starvation ; IP , immunoprecipitation ; a . u . , arbitrary unit . Bar , 10 µm .

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 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 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 O O O O O O O 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 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

Figure 5.(A and B) In vitro translated full-length CIP2A was incubated with purified human PP2A complex and HA-Raptor (A) or HA-S6K1 (B) purified from transiently transfected MCF7 cells. Protein complexes were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting as indicated. Nonspecific IgG served as a control. Note that human reticulocyte (RC) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac.(C) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.(D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.(E) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control (C) or PPP2R1A (R1A) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies. Immunoblots of corresponding cell lysates are shown on the left.(F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right.(G) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full-length CIP2A. Error bars are SDs for four (D) independent experiments or a representative triplicate experiment (C and G).(H) Representative confocal microscopy images of untreated and amino acid-starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.