Figure 2A Confocal laser scanning microscopy analyses in cells treated with BafA1 . Total HA - ATZNNN ( red ) or HA - ATZNNN polymers ( magenta ) in LAMP1 - positive endolysosome ( green ) in WT MEF . ( C ) Same as ( A ) in Cnx - KO MEF . ( D ) Same as ( A ) in WT MEF exposed to kifunensine ( KIF ) . ( E ) Same as ( A ) in WT MEF exposed to Castanospermine ( CST ) . ( F ) Same as ( A ) in sCNX MEF . ( G ) Same as ( A ) in Uggt1 - KO MEF . ( J ) Halo - ATZNNN in Uggt1 - KO MEF mock - transfected ( empty pcDNA3 plasmid ) . ( K , L ) ( K ) Same as ( J ) , with a plasmid for expression of active or ( L ) inactive UGGT1 . ( M ) Quantification of Halo - ATZNNN - positive endolysosomes ( mean , n = 22 , 10 , 15 cells for panels J - L , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 ) .

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Figure 2A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF. (C) Same as (A) in Cnx-KO MEF. (D) Same as (A) in WT MEF exposed to kifunensine (KIF). (E) Same as (A) in WT MEF exposed to Castanospermine (CST). (F) Same as (A) in sCNX MEF. (G) Same as (A) in Uggt1-KO MEF. (J) Halo-ATZNNN in Uggt1-KO MEF mock-transfected (empty pcDNA3 plasmid). (K, L) (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1. (M) Quantification of Halo-ATZNNN-positive endolysosomes (mean, n=22, 10, 15 cells for panels J-L, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001).


Figure 3 ( B - I ) Confocal laser scanning microscopy analyses ( as in Fig . 2 ) in WT MEF treated with BafA1 . ( J , K ) ( J ) Quantification of ATZxxx - positive endolysosomes and ( K ) of ATZxxx polymers - positive endolysosomes ( mean , n = 18 , 30 , 24 , 14 , 27 , 22 , 27 , 18 cells for HA stain and n = 12 , 14 , 14 , 11 , 14 , 14 , 17 , 11 cells for 2C1 stain of panels B - I , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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Figure 3(B-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1. (J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.


Figure 4 ( C ) Co - immunoprecipitation of ATZXXX ( Bait , lower panel ) with CNX ( upper panel ) and BiP ( middle panel ) . Protein content in the total cell extracts ( TCE ) is shown . ( D ) Quantification of ( C ) for CNX association ( n = 3 ) . Unpaired two - tailed t - test , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( E ) Quantification of ( C ) for BiP association ( n = 3 for ATZNNN and ATZQQN ; n = 4 for ATZQNQ and ATZQQQ ) . Unpaired two - tailed t - test , * P < 0 . 005 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( G ) Confocal laser scanning microscopy in Flp - In 3T3 cells stably expressing ATZNNN in the absence of BafA1 ( to allow clearance of misfolded model proteins delivered within endolysosomes ) . Co - localization with Calnexin ( CNX ) . ( H ) Same as ( G ) for BiP co - localization . ( I ) Same as ( G ) for ATZQQQ . ( J ) Same as ( H ) for ATZQQQ . ( K ) HaloTag pulse - chase analysis in MEF monitoring fluorescent Halo - ATZNNN during up to 24 h . ( L ) Same as ( K ) for fluorescent Halo - ATZQQQ . Arrowheads show select clusters of JF646 - labeled Halo - ATZQQQ . Arrowheads show co - localization Halo - ATZQQQ : BiP .

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Figure 4(C) Co-immunoprecipitation of ATZXXX (Bait, lower panel) with CNX (upper panel) and BiP (middle panel). Protein content in the total cell extracts (TCE) is shown. (D) Quantification of (C) for CNX association (n=3). Unpaired two-tailed t-test, ** P<0.01*** P<0.001, **** P<0.0001. (E) Quantification of (C) for BiP association (n=3 for ATZNNN and ATZQQN; n=4 for ATZQNQ and ATZQQQ). Unpaired two-tailed t-test, * P<0.005, ** P<0.01, *** P<0.001, **** P<0.0001. (G) Confocal laser scanning microscopy in Flp-In 3T3 cells stably expressing ATZNNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co-localization with Calnexin (CNX). (H) Same as (G) for BiP co-localization. (I) Same as (G) for ATZQQQ. (J) Same as (H) for ATZQQQ. (K) HaloTag pulse-chase analysis in MEF monitoring fluorescent Halo-ATZNNN during up to 24 h. (L) Same as (K) for fluorescent Halo-ATZQQQ. Arrowheads show select clusters of JF646-labeled Halo-ATZQQQ. Arrowheads show co-localization Halo-ATZQQQ:BiP.


Figure 1The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . Western blot showing the expression of AGR2 dimers ( D ) and monomers ( M ) in HEK293T subjected to DSP - mediated cross - linking and that were previously transfected with either a control siRNA ( siCTL ) or a siRNA targeting AGR2 ( siAGR2 ) for 24 h ( C ) Reduced or non - reduced samples were resolved by SDS - PAGE and immunoblotted using anti - AGR2 , anti - ERK1 antibodies ( for loading control ) . The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . Western blot showing the expression of AGR2 dimers ( D ) and monomers ( M ) in HEK293T subjected to DSP - mediated cross - linking and that were previously transfected with either a control siRNA ( siCTL ) or treated or not with tunicamycin ( Tun ) prior to cross - linking ( D ) . Reduced or non - reduced samples were resolved by SDS - PAGE and immunoblotted using anti - AGR2 anti - calnexin ( CANX ) antibodies ( for loading control ) . The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . F ) AGR2 dimerization was monitored with ERMIT ( wild - type ( WT ) ) . The luminescence signal was abrogated when using the kinase dead ( KD ) constructs and by the constructs exhibiting mutations in the dimerization domain ( E60A or C81S or double mutant ( DM ) ) . The graph represents average signal normalized on reporter protein expression ± SD ( n = 5 ; * * : WT / KD ( p = 0 . 002 ) , E60 / WT ( p = 0 . 0034 ) , E60 / KD ( p = 0 . 001 ) , C81 / KD ( p = 0 . 004 ) , DM / WT ( p = 0 . 0021 ) and DM / KD ( p = 0 . 0017 ) , respectively ; * : C81 / WT ( p = 0 . 0098 ) ) . The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . G ) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT . The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors ( n = 4 ) .

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Figure 1The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or a siRNA targeting AGR2 (siAGR2) for 24 h (C) Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2, anti-ERK1 antibodies (for loading control).The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or treated or not with tunicamycin (Tun) prior to cross-linking (D). Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2 anti-calnexin (CANX) antibodies (for loading control).The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. F) AGR2 dimerization was monitored with ERMIT (wild-type (WT)). The luminescence signal was abrogated when using the kinase dead (KD) constructs and by the constructs exhibiting mutations in the dimerization domain (E60A or C81S or double mutant (DM)). The graph represents average signal normalized on reporter protein expression ± SD (n=5; **: WT/KD (p=0.002), E60/WT (p=0.0034), E60/KD (p=0.001), C81/KD (p=0.004), DM/WT (p=0.0021) and DM/KD (p=0.0017), respectively; *: C81/WT (p=0.0098)).The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. G) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT. The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors (n = 4).


Figure 2B ) Volcano plot representing the statistical analysis of the data from the screen , where the y axis represents statistical criteria and the x axis the fold change in luminescence units .

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Figure 2B) Volcano plot representing the statistical analysis of the data from the screen, where the y axis represents statistical criteria and the x axis the fold change in luminescence units.


Figure 3A ) Co - immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ . * indicates Immunoglobulin heavy and light chains . C ) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells . DSP - stabilized AGR2 was analyzed under non - reducing ( top blot ) or reducing conditions ( bottom blot ) . D = dimeric , M = monomeric AGR2 . E ) : Co - immunoprecipitation of AGR2 wild - type ( WT , left panel ) or AGR2 AA mutant ( right panel ) with TMED2 in HEK293T cells . Immunoprecipitation was performed with mouse anti - Flag antibody to pull - down the ectopic protein tag . F ) Western blot analysis ( left panel ) and quantification ( right panel ) of AGR2 intracellular expression upon TMED2 overexpression . Data are representative of 4 independent experiments . Tubulin ( TUB ) was used as a loading control . G ) HEK293T cells were transfected with control ( ev ) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT ( n = 3 ) . H ) Western blot analysis ( left panel ) and quantification ( right panel ) of AGR2 intracellular expression in total cell lysate ( TCL ) upon TMED2 silencing . Data are representative of 4 independent experiments . Tubulin ( TUB ) was used as a loading control . I ) HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT ( n = 3 ) .

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Figure 3A) Co-immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ. *indicates Immunoglobulin heavy and light chains.C) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP-stabilized AGR2 was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). D=dimeric, M=monomeric AGR2.E): Co-immunoprecipitation of AGR2 wild-type (WT, left panel) or AGR2 AA mutant (right panel) with TMED2 in HEK293T cells. Immunoprecipitation was performed with mouse anti-Flag antibody to pull-down the ectopic protein tag.F) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression upon TMED2 overexpression. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control.G) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT (n=3).H) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression in total cell lysate (TCL) upon TMED2 silencing. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control.I) HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT (n=3).


Figure 4A ) The ERMIT - based validation of the complex formation between AGR2 and LYPD3 or CD59 , but not OS9 . The monomeric AGR2 E60A was used as bait , while LYPD3 , CD59 and OS9 were used as prays ( n = 6 ) . B - C ) Expression and subcellular localization of CD59 - GFP WT and C94S . D and E ) Co - immunoprecipitation of CD59 - GFP WT or CD59 C94S mutant with AGR2 WT ( D ) or AGR2 AA mutant ( E ) in HEK293T cells . F - I ) FACS analysis of CD59 WT ( F and G ) or CD59 C94S ( H and I ) total ( F and H ) and membrane ( G and I ) expression in HEK293T cells silenced for AGR2 ( siAGR2 ) and TMED2 ( siTMED2 ) when compared to control ( siGL2 ) cells ( F and H ) or silenced for TMED2 ( siTMED2 ) and overexpressing AGR2 WT or AGR2 AA mutant ( G and I ) . Data are representative of three to four independent experiments . Membranous CD59 expression was detected with anti - GFP APC . ns : non - statistically significant ; for F ( * ) : p = 0 . 0385 , ( * * * ) : p = 0 . 0003 ( left panel ) and p = 0 . 0002 ( right panel ) , respectively ; for G ( * ) : p = 0 . 0334 , ( * * ) p = 0 . 0036 and ( * * * ) p = 0 . 0003 ; for H ( * ) : p = 0 . 0171 and ( * * ) : p = 0 . 0084 ; and for I ( * * ) p = 0 . 0086 ( left panel ) and p = 0 . 0076 ( right panel ) , respectively .

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Figure 4A) The ERMIT-based validation of the complex formation between AGR2 and LYPD3 or CD59, but not OS9. The monomeric AGR2 E60A was used as bait, while LYPD3, CD59 and OS9 were used as prays (n=6).B-C) Expression and subcellular localization of CD59-GFP WT and C94S.D and E) Co-immunoprecipitation of CD59-GFP WT or CD59 C94S mutant with AGR2 WT (D) or AGR2 AA mutant (E) in HEK293T cells.F-I) FACS analysis of CD59 WT (F and G) or CD59 C94S (H and I) total (F and H) and membrane (G and I) expression in HEK293T cells silenced for AGR2 (siAGR2) and TMED2 (siTMED2) when compared to control (siGL2) cells (F and H) or silenced for TMED2 (siTMED2) and overexpressing AGR2 WT or AGR2 AA mutant (G and I). Data are representative of three to four independent experiments. Membranous CD59 expression was detected with anti-GFP APC. ns: non-statistically significant; for F (*): p=0.0385, (***): p=0.0003 (left panel) and p=0.0002 (right panel), respectively; for G (*): p=0.0334, (**) p=0.0036 and (***) p=0.0003; for H (*): p=0.0171 and (**): p=0.0084; and for I (**) p=0.0086 (left panel) and p=0.0076 (right panel), respectively.


Figure 5A ) Formation of GFP - LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy ( right panel ) . DAPI was used for nuclear staining visualization . Percentage of GFP - LC3 puncta in control ( CTL ) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP - LC3 positive cells for each condition ( left panel ) . ( * * ) : p = 0 . 0032 . B ) Western blot detection ( left panel ) and quantification ( right panel ) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours . Actin ( ACT ) served as a loading control . ( * ) : p = 0 . 0498 . C ) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment . Actin ( ACT ) served as a loading control . D ) HEK293T cells were transfected with control ( ev ) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT the presence of gradual amounts of chloroquine . E ) Western blot analysis of secreted AGR2 upon TMED2 overexpression . eAGR2 , extracellular AGR2 ; iAGE2 , intracellular AGR2 . F ) Representative pictures of extracellular vesicles ( EVs ) heterogeneity purified from conditioned media of HEK293T cells transfected with control ( ev ) or TMED2 plasmid and analyzed by cryo - electron microscopy . Right upper panel : western blot analysis of CD63 in total medium ( left ) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells ( middle ) and western blot analysis of AGR2 levels ( right ) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells . G ) Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2 , extracellular AGR2 ; iAGE2 , intracellular AGR2 . H ) Quantification of extracellular AGR2 level ( eAGR2 ) in cells silenced for TMED2 ( siTMED2 ) , compared to control ( siGL2 ) . Data are representative of three independent experiments and are presented as extracellular - to - intracellular AGR2 ratio . ( * * ) : p = 0 . 0029 . I ) Secretion of AGR2 wild - type ( wt ) , E60A and ∆ 45 mutant as determined by Western blot . eAGR2 , extracellular AGR2 ; iAGE2 , intracellular AGR2 .

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Figure 5A) Formation of GFP-LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy (right panel). DAPI was used for nuclear staining visualization. Percentage of GFP-LC3 puncta in control (CTL) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP-LC3 positive cells for each condition (left panel). (**): p=0.0032.B) Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours. Actin (ACT) served as a loading control. (*): p=0.0498.C) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment. Actin (ACT) served as a loading control.D) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT the presence of gradual amounts of chloroquine.E) Western blot analysis of secreted AGR2 upon TMED2 overexpression. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.F) Representative pictures of extracellular vesicles (EVs) heterogeneity purified from conditioned media of HEK293T cells transfected with control (ev) or TMED2 plasmid and analyzed by cryo-electron microscopy. Right upper panel: western blot analysis of CD63 in total medium (left) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells (middle) and western blot analysis of AGR2 levels (right) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells.G) Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. H) Quantification of extracellular AGR2 level (eAGR2) in cells silenced for TMED2 (siTMED2), compared to control (siGL2). Data are representative of three independent experiments and are presented as extracellular-to-intracellular AGR2 ratio. (**): p=0.0029.I) Secretion of AGR2 wild-type (wt), E60A and ∆45 mutant as determined by Western blot. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.


Figure 6A ) Representative histological H & E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice . B ) Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice . C ) Semi - quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT ( blank bars ) and TMED2 hypomorph mice ( black bars ) ( n = 6 ) . D ) Freshly isolated PBMCs were placed in Boyden chambers towards media conditioned by cells overexpressing AGR2 WT , E60A , ∆ 45 or AGR2 AA mutants and incubated for 24 h . Migrating cells were then characterized and quantified by flow cytometry using FCS / SSC parameters or CD14 monocyte marker . ( * * ) : p = 0 . 0016 . E and F ) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells overexpressing TMED2 and either AGR2 WT ( E ) or AGR2 AA mutant ( F ) and incubated for 24 h . Migrating cells were then characterized and quantified For E ( * ) : p = 0 . 0461 ( AGR2WT / TMED2 ) and 0 . 018 ( AGR2WT / TMED2 + blocking Ab ) respectively ; ( * * ) : p = 0 . 0053 and ( * * * ) : p = 0 . 0048 ; for F ( * * * ) : p = 0 . 0007 . G ) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells silenced for TMED2 and incubated for 24 h . Migrating cells were then characterized and quantified ( * ) : p = 0 . 0149 . H ) Freshly isolated PBMC were placed in Boyden chambers towards decreased doses of recombinant AGR2 and incubated for 24 h . CCL2 cytokine was used as positive control for monocyte migration . ns : non - statistically significant , ( * ) : p = 0 . 0171 , ( * * ) : p = 0 . 0084 , ( * * * ) : p = 0 . 0003 . I ) Impact of AGR2 blocking antibodies on AGR2 - mediated monocytes migration was tested using Boyden chambers as described above . The concentrations of recombinant human AGR2 was of 200 ng / ml and decreasing amounts of antibodies were used from 20 µg to 1 µg . The non - relevant antibody ( Isotype ) was used at the maximal dose of 20 µg . Data are representative of three independent experiments . ns : non - statistically significant , ( * ) : p = 0 . 0165 ( Ab = 20μg / ml ) , 0 . 0223 ( Ab = 10μg / ml ) and 0 . 0493 ( Ab = 5μg / ml ) , respectively .

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Figure 6A) Representative histological H&E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice.B) Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice.C) Semi-quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT (blank bars) and TMED2 hypomorph mice (black bars) (n=6).D) Freshly isolated PBMCs were placed in Boyden chambers towards media conditioned by cells overexpressing AGR2 WT, E60A, ∆45 or AGR2 AA mutants and incubated for 24 h. Migrating cells were then characterized and quantified by flow cytometry using FCS/SSC parameters or CD14 monocyte marker. (**): p=0.0016.E and F) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells overexpressing TMED2 and either AGR2 WT (E) or AGR2 AA mutant (F) and incubated for 24 h. Migrating cells were then characterized and quantified For E (*): p=0.0461 (AGR2WT/TMED2) and 0.018 (AGR2WT/TMED2 + blocking Ab) respectively; (**): p=0.0053 and (***): p=0.0048; for F (***): p=0.0007.G) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells silenced for TMED2 and incubated for 24 h. Migrating cells were then characterized and quantified (*): p=0.0149.H) Freshly isolated PBMC were placed in Boyden chambers towards decreased doses of recombinant AGR2 and incubated for 24 h. CCL2 cytokine was used as positive control for monocyte migration. ns: non-statistically significant, (*): p=0.0171, (**): p=0.0084, (***): p=0.0003.I) Impact of AGR2 blocking antibodies on AGR2-mediated monocytes migration was tested using Boyden chambers as described above. The concentrations of recombinant human AGR2 was of 200 ng/ml and decreasing amounts of antibodies were used from 20 µg to 1 µg. The non-relevant antibody (Isotype) was used at the maximal dose of 20 µg. Data are representative of three independent experiments. ns: non-statistically significant, (*): p=0.0165 (Ab=20μg/ml), 0.0223 (Ab=10μg/ml) and 0.0493 (Ab=5μg/ml), respectively.


Figure 7A ) Selective enrichment of AGR2 modulators in colonic Crohn ' s disease ( CD ) . Percentage of tested genes with altered expression in colonic biopsies from patients with Ulcerative Colitis ( UC , n = 8 ) and colonic CD ( CC , n = 15 ) and in ileal biopsies from patients with ileo - colonic CD ( IC , n = 9 ) . B ) Hierarchical clustering of the AGR2 modulator genes significantly deregulated in non - inflamed colonic mucosa from patients with Crohn ' s Disease ( CD ) versus healthy controls ( C ) ( dChip software t - Test ) C ) TMED2 mRNA expression in healthy controls ( CT ) , colonic Crohn ' s disease ( CC ) and ulcerative colitis ( UC ) . D ) Representative immunohistological analysis of TMED2 in non - inflamed colonic biopsies from healthy controls and patients with active CD . E ) Representative immunohistological analysis of CD163 ( scavenger receptor present in macrophages ) , AGR2 , and TMED2 in non - inflamed colonic biopsies from healthy controls and patients with active or quiescent CD . F ) Semi - quantitative analysis of CD163 , AGR2 , and TMED2 immunostaining in colonic mucosa from healthy controls ( C ) and patients with quiescent ( Q ) or active ( A ) CD . Three random sections from each patient of the four groups were scored . Final IHC scores ( AxB range to 0 - 9 ) combine the percentage of positively labelled cells . A : % of IHC positive labeled cells - 0 ( 0 % ) , 1 ( < 30 % ) , 2 ( 30 to 60 % ) , 3 ( > 60 % ) ) and the intensity of the reaction product . B : 0 ( no reaction ) , 1 ( weak ) , 2 ( mild ) , 3 ( strong ) in most of the examined fields . All values are mean ± S . E . M .

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Figure 7A) Selective enrichment of AGR2 modulators in colonic Crohn's disease (CD). Percentage of tested genes with altered expression in colonic biopsies from patients with Ulcerative Colitis (UC, n=8) and colonic CD (CC, n=15) and in ileal biopsies from patients with ileo-colonic CD (IC, n=9).B) Hierarchical clustering of the AGR2 modulator genes significantly deregulated in non-inflamed colonic mucosa from patients with Crohn's Disease (CD) versus healthy controls (C) (dChip software t-Test)C) TMED2 mRNA expression in healthy controls (CT), colonic Crohn's disease (CC) and ulcerative colitis (UC).D) Representative immunohistological analysis of TMED2 in non-inflamed colonic biopsies from healthy controls and patients with active CD.E) Representative immunohistological analysis of CD163 (scavenger receptor present in macrophages), AGR2, and TMED2 in non-inflamed colonic biopsies from healthy controls and patients with active or quiescent CD.F) Semi-quantitative analysis of CD163, AGR2, and TMED2 immunostaining in colonic mucosa from healthy controls (C) and patients with quiescent (Q) or active (A) CD. Three random sections from each patient of the four groups were scored. Final IHC scores (AxB range to 0-9) combine the percentage of positively labelled cells. A: % of IHC positive labeled cells - 0 (0%), 1 (<30%), 2 (30 to 60%), 3 (>60%)) and the intensity of the reaction product. B: 0 (no reaction), 1 (weak), 2 (mild), 3 (strong) in most of the examined fields. All values are mean ± S.E.M.


Figure 1 ( A ) Quantification of CXCR4 + cells by flow cytometry and Sox17 + cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH . Average and standard deviation ( SD ) of eight independent experiments . ( B ) Gene expression profiling by RT - PCR at day 7 differentiation . Average and SD of three independent experiments . ( C ) Immunostaining for Ecadherin ( left ) and FoxA2 ( right ) at day 7 of differentiation in presence of 3 µM CH . ( D ) Quantification of CXCR4 + by flow cytometry over a seven - day differentiation time course in the presence of 3 µM CH . Average and SD of three independent experiments . ( E ) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH . Scale bars = 200

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Figure 1(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments.(B) Gene expression profiling by RT-PCR at day 7 differentiation. Average and SD of three independent experiments.(C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH.(D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments.(E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM


Figure 2 ( A ) Assay of Tcf7l1 mRNA by RT - PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH . Average and SD of three independent experiments . Day2 * p = 0 . 048 , Day3 * p = 0 . 036 . B ) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence ( + ) or absence ( - ) of 3 μM CH . ( C ) Assay of FoxA2 mRNA by RT - PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . * * p = 5x10 - 7 . ( D ) ChiP - seq data from a compendium of transcription factor ChIP - seq analyses ( Sánchez - Castillo et al , 2015 ) . Gene tracks represent binding of Tcf7l1 at the FoxA2 , Nodal and Eomesodermin ( Eomes ) gene locus . Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1 . ( E ) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig . 2D . Analysis was performed in wild - type ES cells in 2i culture conditions . Average and SD of three independent experiments . Klf2 * p = 0 . 025 , FoxA2 * p = 0 . 050 , Eomes * p = 0 . 023 . ( F ) ChIP for Tcf7l1 followed by qPCR . Analysis was performed in wild - type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . FoxA2 * p = 0 . 049 .

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Figure 2(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.B) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence (+) or absence (-) of 3 μM CH.(C) Assay of FoxA2 mRNA by RT-PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. **p=5x10-7.(D) ChiP-seq data from a compendium of transcription factor ChIP-seq analyses (Sánchez-Castillo et al, 2015). Gene tracks represent binding of Tcf7l1 at the FoxA2, Nodal and Eomesodermin (Eomes) gene locus. Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1.(E) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig. 2D. Analysis was performed in wild-type ES cells in 2i culture conditions. Average and SD of three independent experiments. Klf2 *p=0.025, FoxA2 *p=0.050, Eomes *p=0.023.(F) ChIP for Tcf7l1 followed by qPCR. Analysis was performed in wild-type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. FoxA2 *p = 0.049.


Figure 3 ( A ) Assay of FoxA2 mRNA by RT - PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH . Average and SD of three independent experiments . ( B ) Gene expression analysis of endodermal associated genes by RT - PCR in control wild - type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments . ( C ) FoxA2 ( green ) and Oct4 ( red ) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH . DAPI staining in blue . ( D ) Assay of CXCR4 , Hex and Sox17 mRNA by RT - PCR in control wild - type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments .

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Figure 3(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments.(B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.(C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue.(D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.


Figure 4 ( A ) Assay of FoxA2 mRNA by RT - PCR in FoxA2 - Tet ES cell lines 48 hr after treatment with the indicated dose of DOX . Average and SD of two independent experiments from 2 independent lines . B ) FoxA2 immunostaining of FoxA2 - Tet ES cells 48 hr after DOX treatment . Left , no DOX control . Middle and right , 10ng / ml DOX . DAPI staining shown in blue . Right panel shows middle image without DAPI signal . ( C ) Assay of FoxA2 and CXCR4 mRNA by RT - PCR on day 3 of differentiation with DOX at the indicated dose . Average and SD of two experiments from two independent cell lines . ( D ) Assay of Sox17 mRNA by RT - PCR on day 7 of differentiation with addition of 3 µM CH and / or 10 ng / ml DOX at the indicated time points ( in days ) . Average and SD of two independent experiments from two independent cell lines . ( E ) Sox17 immunostaining of FoxA2 - Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng / ml DOX and the presence and absence of 3 µM CH as indicated . Far right panel shows image to its left without DAPI .

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Figure 4(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines.B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal.(C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines.(D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines.(E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.


Figure 5 ( A ) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng / ml ( W100 ) or 200 ng / ml ( W200 ) recombinant Wnt3a plus 500 ng / ml recombinant RSpondin1 . Relative luciferase units are normalised to FOPflash . Average and SD of three independent experiments . ( B ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a ( 200 ng / ml plus 500 ng / ml RSpondin1 ( WR ) ) . Average and SD of three independent experiments . * * p < 0 . 01 . ( C ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH . Average and SD of three independent experiments . ( D ) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH . Results from two independent , clonally - derived Ctnnb1 null ES cell lines are shown . Tubulin was used as a loading control .

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Figure 5(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments.(B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01.(C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments.(D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. Tubulin was used as a loading control.


Figure 6 ( A ) Assay of cMyc mRNA by RT - PCR in wild - type ES cells at day 1 to 3 of differentiation . Average and SD of three independent experiments . ( B ) Immunoblot for total cMyc and phospho Ser62 ( pSer62 ) cMyc during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH . C , D ) Immunoblot for the destabilized form of cMyc protein ( pThr58 ) during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH in wild - type and Ctnnb1 null cells respectively . C , D ) Immunoblot for the destabilized form of cMyc protein ( pThr58 ) during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH in wild - type and Ctnnb1 null cells respectively . ( E ) Assay of Tcf7l1 mRNA by RT - PCR in wild - type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc ( cMyc Inhibitor II ; Myci ) . Treatment with Myc inhibitor 10058 - F4 ( Sigma ) generated similar results ( data not shown ) . Average and SD of three independent experiments . * * p = 0 . 0002 . ( F ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in cMyc null / nMyc heterozygous cells ( C - / - ; N + / - ) and floxed parental cells ( cMycfl / fl ; nMycfl / fl ( control ) ) at day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of four or six independent experiments . Tcf7l1 * p = 0 . 01 , FoxA2 * p = 0 . 034 . ( G ) FoxA2 immunostaining of control and cMyc null / nMyc heterozygous ( cMyc - / - ; nMyc + / - ) cells at day 3 of differentiation in the presence of 3 µM CH . ( H ) Assay of cMyc and Tcf7l1 mRNA by RT - PCR in cMycT58A inducible cell lines ( icMycT58A ) 24 hr after DOX treatment . Average of 2 independent experiments . ( I ) Assay of Tcf7l1 mRNA by RT - PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng / ml DOX . Average and SD of 3 independent experiments . * * p = 0 . 007 . ( J ) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng / ml .

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Figure 6(A) Assay of cMyc mRNA by RT-PCR in wild-type ES cells at day 1 to 3 of differentiation. Average and SD of three independent experiments.(B) Immunoblot for total cMyc and phospho Ser62 (pSer62) cMyc during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH.C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.(E) Assay of Tcf7l1 mRNA by RT-PCR in wild-type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc (cMyc Inhibitor II; Myci). Treatment with Myc inhibitor 10058-F4 (Sigma) generated similar results (data not shown). Average and SD of three independent experiments. **p=0.0002.(F) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in cMyc null/nMyc heterozygous cells (C-/-;N+/-) and floxed parental cells (cMycfl/fl;nMycfl/fl (control)) at day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of four or six independent experiments. Tcf7l1 *p=0.01, FoxA2 *p=0.034.(G) FoxA2 immunostaining of control and cMyc null/nMyc heterozygous (cMyc-/-;nMyc+/-) cells at day 3 of differentiation in the presence of 3 µM CH.(H) Assay of cMyc and Tcf7l1 mRNA by RT-PCR in cMycT58A inducible cell lines (icMycT58A) 24 hr after DOX treatment. Average of 2 independent experiments.(I) Assay of Tcf7l1 mRNA by RT-PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng/ml DOX. Average and SD of 3 independent experiments. **p=0.007.(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.


Figure 7 ( B ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the absence or presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * p = 0 . 028 . ( C ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058 - F4 . Average and SD for three independent experiments , * p = 0 . 026 . ( D ) Assay for Miz1 mRNA by RT - PCR for the first three days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . ( E ) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * * p = 0 . 0015 .

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 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

Figure 7(B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028.(C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026.(D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments.(E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.


Figure 1 WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while the basal bodies were marked with C - Nap1 ( red ) . DNA was stained with DAPI . Cilia in the magenta box are shown enlarged on the right picture WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while the basal bodies were marked with C - Nap1 ( red ) . DNA was stained with DAPI Percent of ciliated cells , as well as the length of cilia , was quantified from A . Cilia length was measured by a semi - automated ImageJ macro as described in methods . Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition . Error bars represent the mean ± SD . * * * * P ≤ 0 . 0001 . High means 80 - 90 % and low 40 - 50 % confluency of RPE1 cells Elongated cilia phenotype was confirmed by siRNA - mediated knockdown of hCDC14A . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * * * * P ≤ 0 . 0001 hCDC14A - GFP and hCDC14AC278S - GFP under control of the TetON promoter were stably integrated into RPE1 cells . hCDC14A - GFP and hCDC14AC278S - GFP localized to the C - Nap1 marked basal body upon Dox induction . In addition , expression of hCDC14A - GFP but not the phosphatase dead hCDC14AC278S - GFP mutant inhibited cilia formation . Ciliated cells were quantified 48 h after serum starvation with and without Dox addition . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * * P ≤ 0 . 001 ; ns : not significant Expression of hCDC14A - GFP and hCDC14AC278S - GFP was validated by immunoblot analysis with anti - GFP antibodies . GAPDH was the loading control RPE1 cells over - expressing hCDC14AC278S have longer cilia than those over - expressing hCDC14A . Three independent experiments , N = 100 cilia for hCDC14A expressing cells and 200 for hCDC14AC278S expressing cells . Mean ± SD . * * * P ≤ 0 . 001 The dynamics of ciliogenesis was determined by serum starving WT and hCDC14APD cells for different time - points . The cilia from hCDC14APD cells continue to elongate even after 48 h of starvation whereas those from WT cells reached constant length within 12 to 24 h . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SEM . * P ≤ 0 . 05 , * * ; P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 In a time course experiment , the cilia disassembly kinetics was measured by first inducing ciliation through 48 h of serum starvation prior to incubation with serum containing media for different time points . Measurement of cilia length indicated the comparable rates of cilia disassembly in RPE1 WT and hCDC14APD cells . Cilia disassembly rates are shown in Fig . EV1D . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SEM . * * * * P < 0 . 0001 Images of cilia of WT RPE1 and hCDC14APD RPE1 cells from I , 0 , 6 and 24 h after serum addition . Fixed cells were analyzed for cilia length

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI. Cilia in the magenta box are shown enlarged on the right picture  WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI Percent of ciliated cells, as well as the length of cilia, was quantified from A. Cilia length was measured by a semi-automated ImageJ macro as described in methods. Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition. Error bars represent the mean ± SD. **** P≤ 0.0001. High means 80-90% and low 40-50% confluency of RPE1 cells Elongated cilia phenotype was confirmed by siRNA-mediated knockdown of hCDC14A. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001  hCDC14A-GFP and hCDC14AC278S-GFP under control of the TetON promoter were stably integrated into RPE1 cells. hCDC14A-GFP and hCDC14AC278S-GFP localized to the C-Nap1 marked basal body upon Dox induction. In addition, expression of hCDC14A-GFP but not the phosphatase dead hCDC14AC278S-GFP mutant inhibited cilia formation. Ciliated cells were quantified 48 h after serum starvation with and without Dox addition. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. *** P≤ 0.001; ns: not significant  Expression of hCDC14A-GFP and hCDC14AC278S-GFP was validated by immunoblot analysis with anti-GFP antibodies. GAPDH was the loading control  RPE1 cells over-expressing hCDC14AC278S have longer cilia than those over-expressing hCDC14A. Three independent experiments, N = 100 cilia for hCDC14A expressing cells and 200 for hCDC14AC278S expressing cells. Mean ± SD. *** P≤ 0.001  The dynamics of ciliogenesis was determined by serum starving WT and hCDC14APD cells for different time-points. The cilia from hCDC14APD cells continue to elongate even after 48 h of starvation whereas those from WT cells reached constant length within 12 to 24 h. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SEM. * P≤ 0.05, **; P≤ 0.01; **** P≤ 0.0001 In a time course experiment, the cilia disassembly kinetics was measured by first inducing ciliation through 48 h of serum starvation prior to incubation with serum containing media for different time points. Measurement of cilia length indicated the comparable rates of cilia disassembly in RPE1 WT and hCDC14APD cells. Cilia disassembly rates are shown in Fig. EV1D. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SEM. **** P<0.0001  Images of cilia of WT RPE1 and hCDC14APD RPE1 cells from I, 0, 6 and 24 h after serum addition. Fixed cells were analyzed for cilia length


Figure 2TetON - hCDC14A - GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy . hCDC14A - GFP colocalizes with Phalloidin 555 conjugated dye that marks F - actin fibers . The boxed area was 10 - fold enlarged and displayed below the corresponding images to indicate co - localization of hCDC14A - GFP and F - actin . Line scan inside the magenta colored box showed in ( A ) Sub - centrosomal localization of hCDC14AC278S - GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 ( cilia ) , C - Nap1 / ninein ( proximal end ) , CEP164 ( distal appendage ) and ODF2 / ninein ( subdistal appendage ) . Results are fitted in the cartoons adjacent to the respective panels for better

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Figure 2TetON-hCDC14A-GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy. hCDC14A-GFP colocalizes with Phalloidin 555 conjugated dye that marks F-actin fibers. The boxed area was 10-fold enlarged and displayed below the corresponding images to indicate co-localization of hCDC14A-GFP and F-actin. Line scan inside the magenta colored box showed in (A)Sub-centrosomal localization of hCDC14AC278S-GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 (cilia), C-Nap1/ninein (proximal end), CEP164 (distal appendage) and ODF2/ ninein (subdistal appendage). Results are fitted in the cartoons adjacent to the respective panels for better visualization


Figure 4hCDC14A , not the inactive hCDC14AC278S , dephosphorylates the serine residue 142 of DBN1 in vivo . RPE1 cells expressing GFP , hCDC14A - GFP , and hCDC14AC278S - GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence . DBN1pS142 was detected with a phospho - specific antibody . DNA was stained with DAPI . Scale Bar = 10 μmImmunoblot analysis of samples from ( A ) . The DBN1pS142 / DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot . In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots . Three independent experiments . Mean ± SD . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001DBN1S142 was hyper - phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control . In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot . The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock - down of DBN1 in both hCDC14APD cells and GFP / hCDC14A - GFP / hCDC14AC278S - GFP stable cell lines . Three independent experiments . Mean ± SD . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001In vitro dephosphorylation of DBN1 by purified hCDC14A . DBN1 - GFP was transfected into HEK293T cells and expressed for 48 h . DBN1 - GFP was immunoprecipitated from cell lysates with GFP binder . The immunoprecipitated phospho - DBN1 was then incubated with and without recombinant hCDC14A in vitro . The DBN1pS142 / DBN1 ratio was densitometrically measured . Three independent experiments . Mean ± SD . * * * * P ≤ 0 .

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Figure 4hCDC14A, not the inactive hCDC14AC278S, dephosphorylates the serine residue 142 of DBN1 in vivo. RPE1 cells expressing GFP, hCDC14A-GFP, and hCDC14AC278S-GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence. DBN1pS142 was detected with a phospho-specific antibody. DNA was stained with DAPI. Scale Bar = 10 μmImmunoblot analysis of samples from (A). The DBN1pS142/DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot. In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001DBN1S142 was hyper-phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control. In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot. The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock-down of DBN1 in both hCDC14APD cells and GFP/hCDC14A-GFP/hCDC14AC278S-GFP stable cell lines. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001In vitro dephosphorylation of DBN1 by purified hCDC14A. DBN1-GFP was transfected into HEK293T cells and expressed for 48 h. DBN1-GFP was immunoprecipitated from cell lysates with GFP binder. The immunoprecipitated phospho-DBN1 was then incubated with and without recombinant hCDC14A in vitro. The DBN1pS142/DBN1 ratio was densitometrically measured. Three independent experiments. Mean ± SD. **** P≤ 0.0001


Figure 5The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate . GAPDH is the loading control . One targeted clone from each gRNA ( 1 . 16 , 2 . 18 , 3 . 3 ) including the non - targeted clone 2 . 19 and WT control were serum starved for 48 h to induce ciliogenesis . Subsequently , cilia length was measured . The cilia from DBN1 KO cells were significantly longer than those from WT or non - targeted controls . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * * * P ≤ 0 . 0001 . Overexpression of DBN1 , DBN1S142A and DBN1S142D in RPE1 WT cells . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; ns : not specific . The phospho - inhibitory ( S142A ) version of DBN1 rescued the elongated cilia of DBN1 KO cells . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 ; ns : not significant . Pairwise comparison was done with unpaired student ' s t - test and the p - value was written in the figure . Global comparison was done with one - way ANOVA and the p - values were marked with stars . The overexpressed WT DBN1 was hyper - phosphorylated in cells as detected by phospho - specific DBN1pS142 antibodies . The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D . Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo - phosphorylation of DBN1S142 in response to CDK5 depletion The DBN1pS142 / GAPDH ratio from ( H ) was densitometrically measured . Three independent experiments . Mean ± SD . * P ≤ 0 . 05 Depletion of CDK5 decreased the cilia length in WT cells . It restored cilia length in hCDC14APD cells . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * ; P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 ; ns : not significant . CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho - mimetic DBN1S142D in hCDC14APD cells . Three independent experiments ; 150 cells per experiment and condition . Mean ± SD ; ns = not significant ; * * * * P ≤ 0 . 0001 .

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

Figure 5The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate. GAPDH is the loading control.One targeted clone from each gRNA (1.16, 2.18, 3.3) including the non-targeted clone 2.19 and WT control were serum starved for 48 h to induce ciliogenesis. Subsequently, cilia length was measured. The cilia from DBN1 KO cells were significantly longer than those from WT or non-targeted controls. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001.Overexpression of DBN1, DBN1S142A and DBN1S142D in RPE1 WT cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. ** P≤ 0.01; *** P≤ 0.001; ns: not specific. The phospho-inhibitory (S142A) version of DBN1 rescued the elongated cilia of DBN1 KO cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. * P≤ 0.05; *** P≤ 0.001; ns: not significant. Pairwise comparison was done with unpaired student's t-test and the p-value was written in the figure. Global comparison was done with one-way ANOVA and the p-values were marked with stars.  The overexpressed WT DBN1 was hyper-phosphorylated in cells as detected by phospho-specific DBN1pS142 antibodies. The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D.  Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo-phosphorylation of DBN1S142 in response to CDK5 depletion The DBN1pS142/GAPDH ratio from (H) was densitometrically measured. Three independent experiments. Mean ± SD. * P≤ 0.05 Depletion of CDK5 decreased the cilia length in WT cells. It restored cilia length in hCDC14APD cells. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **; P≤ 0.01; **** P≤ 0.0001; ns: not significant.CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho-mimetic DBN1S142D in hCDC14APD cells. Three independent experiments; 150 cells per experiment and condition. Mean ± SD; ns = not significant; **** P≤ 0.0001.


Figure 6 Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [ 17 ] . Fluorescent transferrin was added for 45 min to the cells . Cells were analyzed after washing . The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for ( B ) . The encircled area in MERGE is shown 2 - fold enlarged on the right hand side of the figure Quantification of ( A ) . The fluorescence intensities of transferrin within a 2 μm - radius circle surrounding the CEP164 signal were quantified . An ImageJ macro was used to measure the intensity as described in methods . Data were calculated from a total of 120 cells pooled from two independent experiments . Mean ± SD . * * * * P ≤ 0 . 0001 hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm - radius of the basal body ( red cicle ) . The encircled area in MERGE is shown 4 - fold enlarged on the right hand side of the figure Quantification of C . Data was calculated from a total of 180 cells pooled from three independent experiments . Mean ± SD . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 Arp2 localization in REP1 WT , hCDC14APD and DBN1 KO cells . Signal intensity was measured 30 min after serum starvation . The encircled area in MERGE is shown 4 - fold enlarged on the right hand side of the figure Quantification of E . Sub - centrosomal ( 1 μm - radius ; red circle in E ) Arp2 was measured by quantifying the intensity of Arp2 in WT , hCDC14APD and DBN1 KO cells . Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area ( 1 μm - radius ) compared to WT cells after 30 min of serum starvation . Data were calculated from a total of 120 cells pooled from three independent experiments . Mean ± SD . * * * * P ≤ 0 . 0001 Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells ( Tet Control ) with DBN1 KO cells expressing different DBN1 versions ( WT , S142A , S142D ) . Data were calculated from a total of 200 cells each pooled from two independent experiments . Mean ± SD . ns : not specific

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [17]. Fluorescent transferrin was added for 45 min to the cells. Cells were analyzed after washing. The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for (B). The encircled area in MERGE is shown 2-fold enlarged on the right hand side of the figure Quantification of (A). The fluorescence intensities of transferrin within a 2 μm-radius circle surrounding the CEP164 signal were quantified. An ImageJ macro was used to measure the intensity as described in methods. Data were calculated from a total of 120 cells pooled from two independent experiments. Mean ± SD. **** P≤ 0.0001  hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm-radius of the basal body (red cicle). The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure Quantification of C. Data was calculated from a total of 180 cells pooled from three independent experiments. Mean ± SD. * P≤ 0.05, *** P≤ 0.001, **** P≤ 0.0001  Arp2 localization in REP1 WT, hCDC14APD and DBN1 KO cells. Signal intensity was measured 30 min after serum starvation. The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure  Quantification of E. Sub-centrosomal (1 μm-radius; red circle in E) Arp2 was measured by quantifying the intensity of Arp2 in WT, hCDC14APD and DBN1 KO cells. Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area (1 μm-radius) compared to WT cells after 30 min of serum starvation. Data were calculated from a total of 120 cells pooled from three independent experiments. Mean ± SD. **** P≤ 0.0001  Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells (Tet Control) with DBN1 KO cells expressing different DBN1 versions (WT, S142A, S142D). Data were calculated from a total of 200 cells each pooled from two independent experiments. Mean ± SD. ns: not specific


Figure 1 ( A ) CXCL10 assay characteristics . Standard curves of the short , long and total CXCL10Simoa assays are shown . For each curve , limit of detection ( LOD ) defined as blank + 3SD , are shown as horizontal lines . The LOD was 1 . 7pg / ml for long and short CXCL10 assays and 0 . 22pg / ml for total CXCL10 . Standard curves were fitted using the 4 Parameter Logistic non - linear regression model . Samples reporting a signal below the LOD were replaced with 1pg / ml for short and long CXCL10 . Plasma from healthy individuals receiving ( B ) placebo ( n = 9 ) or ( C ) sitagliptin ( n = 27 ) were analyzed by Simoa . Blood samples were collected at screening visit ( SV ) and day 0 ( D0 ) before treatment , at day 3 ( D3 ) , day 14 ( W2 ) and day 28 ( W4 ) under treatment and 5 weeks after treatment interruption ( W9 ) . ( C ) Plasma from chronic HCVpatients receiving sitagliptin ( n = 3 ) were collected before ( D0 ) and weekly during sitagliptin treatment ( W1 W3 ) . Antagonist CXCL103 - 77 ( short CXCL10 , in blue ) , agonist CXCL101 - 77 ( long CXCL10 , in red ) and total CXCL10 ( in black ) levels are shown . Each dot represents a donor and bars are at the median . Grey areas highlight the period under placebo or sitagliptin treatment . Statistical analysis of ( B ) and ( C ) was performed using non parametric Friedman ' s test , ns : non significant , * * : P0 . 01 , * * * * : P0 . 001 . For ( C ) , additional size effect analysis was performed and Cohen ' s d values are reported . No statistical analysis was performed in ( D ) due to sample size , nd : non - determined .

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Figure 1(A) CXCL10 assay characteristics. Standard curves of the short, long and total CXCL10Simoa assays are shown. For each curve, limit of detection (LOD) defined as blank+3SD, are shown as horizontal lines. The LOD was 1.7pg/ml for long and short CXCL10 assays and 0.22pg/ml for total CXCL10. Standard curves were fitted using the 4 Parameter Logistic non-linear regression model. Samples reporting a signal below the LOD were replaced with 1pg/ml for short and long CXCL10.Plasma from healthy individuals receiving (B) placebo (n=9) or (C) sitagliptin (n=27) were analyzed by Simoa. Blood samples were collected at screening visit (SV) and day 0 (D0) before treatment, at day 3 (D3), day 14 (W2) and day 28 (W4) under treatment and 5 weeks after treatment interruption (W9). (C) Plasma from chronic HCVpatients receiving sitagliptin (n=3) were collected before (D0) and weekly during sitagliptin treatment (W1 W3). Antagonist CXCL103-77 (short CXCL10, in blue), agonist CXCL101-77 (long CXCL10, in red) and total CXCL10 (in black) levels are shown. Each dot represents a donor and bars are at the median. Grey areas highlight the period under placebo or sitagliptin treatment. Statistical analysis of (B) and (C) was performed using non parametric Friedman's test, ns: non significant, **: P0.01, ****: P0.001. For (C), additional size effect analysis was performed and Cohen's d values are reported. No statistical analysis was performed in (D) due to sample size, nd: non-determined.


Figure 1 ( D ) Two - color dSTORM imaging of RIM1 / 2 and gephyrin in cryosections . From left to right : rendered images of RIM1 / 2 and gephyrin clusters showing the aligned SSDs ( arrows ) ; gephyrin SSDs segmented by watershed outlined with different colors ; RIM1 / 2 SSDs outlined with different colors ; binary SSDs of gephyrin and RIM1 / 2 . Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images . Scale bar : 200 nm . RIM1 / 2 was labelled with Alexa 647 , gephyrin ( mAb7a ) with Cy3B in these experiments .

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Figure 1(D) Two-color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labelled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments.


Figure 2 ( A - C ) Alignment of SSDs of pre - synaptic RIM1 / 2 and SSDs of post - synaptic gephyrin , GlyRs , and GABAARs , respectively . ( A1 - C1 ) Distances were measured between the intensity peaks of paired SSDs in rendered dual - color dSTORM images ( yellow line , values given in mean ± SD ) . RIM1 / 2 was labelled with Alexa 647 and gephyrin ( mAb7a ) with Cy3B in ( A1 ) . RIM1 / 2 was labelled with Cy3B , while GlyRs or GABAARs were labelled with Alexa 647 in ( B1 ) and ( C1 ) . ( A2 - C2 ) Trans - synaptic alignment of SSDs of gephyrin , GlyRs , GABAARs and RIM1 / 2 . SSDs were segmented by H - watershed . Data are plotted as mean ± SD . Number of synapses : n = 48 ( A1 , A2 ) from three independent experiments ( no treatment ) , n = 30 ( B1 , B2 ) from two experiments with TTX treatment , n = 16 ( C1 , C2 ) from two experiments with TTX . Only synapses with side view profiles were included . All images are adjusted to the same scale . Scale bar : 200 nm .

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Figure 2(A-C) Alignment of SSDs of pre-synaptic RIM1/2 and SSDs of post-synaptic gephyrin, GlyRs, and GABAARs, respectively. (A1-C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual-color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labelled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labelled with Cy3B, while GlyRs or GABAARs were labelled with Alexa 647 in (B1) and (C1). (A2-C2) Trans-synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs and RIM1/2. SSDs were segmented by H-watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm.


Figure 3 ( A - B ) Representative dSTORM images of GlyRs and GABAARs at synapses in side view ( A ) or en face view ( B ) . The top row of images shows the pointillist representation of the detections , the second row the rendered images and the bottom row the corresponding segmented SSDs . GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments . Scale bar : 200 nm .

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Figure 3(A-B) Representative dSTORM images of GlyRs and GABAARs at synapses in side view (A) or en face view (B). The top row of images shows the pointillist representation of the detections, the second row the rendered images and the bottom row the corresponding segmented SSDs. GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments. Scale bar: 200 nm.


Figure 4 ( A ) Triple immuno - labelling of gephyrin , GlyRs and GABAARs at mixed inhibitory synapses ( arrow ) imaged with conventional fluorescence microscopy . Scale bar : 5 µm .

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Figure 4(A) Triple immuno-labelling of gephyrin, GlyRs and GABAARs at mixed inhibitory synapses (arrow) imaged with conventional fluorescence microscopy. Scale bar: 5 µm.


Figure 5Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses . ( A ) Synaptic receptor cluster rendered from detections of both GABAARs ( labelled with Alexa 647 ) and GlyRs ( labelled with Cy3B ) . ( B ) Detections of GABAARs ( red dots ) and GlyRs ( blue dots ) overlaid with binary synaptic mask ( white ) . Sub - synaptic characterization of GABAAR and GlyR domains . ( F ) Watershed segmentation of sub - synaptic domains ( SSDs ) . Each SSD is shown in different colors . ( G ) Detections of GABAARs ( red dots ) and GlyRs ( blue dots ) overlaid with SSDs ( white ) .

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Figure 5Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (A) Synaptic receptor cluster rendered from detections of both GABAARs (labelled with Alexa 647) and GlyRs (labelled with Cy3B). (B) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with binary synaptic mask (white).Sub-synaptic characterization of GABAAR and GlyR domains. (F) Watershed segmentation of sub-synaptic domains (SSDs). Each SSD is shown in different colors. (G) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with SSDs (white).


Figure 6 ( A ) The number of receptor SSDs , segmented from the combined receptor clusters of GABAARs and GlyRs , was decreased after 4 - AP treatment ( MW test , p < 0 . 0001 ) . ( B ) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only ( MW , * * * * p < 0 . 0001 ) . ( C ) The number of GlyR SSDs ( based on GlyR detections ) were only marginally reduced ( MW , * * p < 0 . 01 ) . Data information : Number of synapses : n = 452 in TTX , n = 583 in 4 - AP condition , from three independent experiments . Mean ± SEM .

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Figure 6(A) The number of receptor SSDs, segmented from the combined receptor clusters of GABAARs and GlyRs, was decreased after 4-AP treatment (MW test, p < 0.0001). (B) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only (MW, ****p < 0.0001). (C) The number of GlyR SSDs (based on GlyR detections) were only marginally reduced (MW, **p < 0.01). Data information: Number of synapses: n = 452 in TTX, n = 583 in 4-AP condition, from three independent experiments. Mean ± SEM.


Figure 7 ( A - C ) Reduced immuno - reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4 - AP treatment , revealed by conventional fluorescence microscopy ( KS test , p < 0 . 001 in B1 and p < 0 . 0001 in B2 , p < 0 . 0001 in C ) . Total gephyrin was probed with polyclonal rabbit primary antibody ( rbGPHN ) , and pS270 phosphorylated gephyrin with monoclonal mouse primary antibody ( m7a ) . Number of synapses : n = 4040 in TTX and n = 3818 in 4 - AP conditions from two independent experiments . Scale bar : 2 µm . ( D - F ) Reduced numbers of pS270 gephyrin ( m7a ) detections were recorded by dSTORM for the entire synaptic area ( D , KS test , p < 0 . 01 ) . The number of detections of pS270 gephyrin per SSD ( E , KS , p = 0 . 18 ) and the number of SSDs ( F , MW , p = 0 . 11 ) were not changed by 4 - AP treatment . Gephyrin was probed with mAb7a antibody and Alexa 647 dye . Number of synapses : n = 810 in TTX and n = 727 in 4 - AP conditions from three independent experiments ; ( F ) mean ± SEM . Scale bar : 100 nm .

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Figure 7(A-C) Reduced immuno-reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4-AP treatment, revealed by conventional fluorescence microscopy (KS test, p < 0.001 in B1 and p < 0.0001 in B2, p < 0.0001 in C). Total gephyrin was probed with polyclonal rabbit primary antibody (rbGPHN), and pS270 phosphorylated gephyrin with monoclonal mouse primary antibody (m7a). Number of synapses: n = 4040 in TTX and n = 3818 in 4-AP conditions from two independent experiments. Scale bar: 2 µm.(D-F) Reduced numbers of pS270 gephyrin (m7a) detections were recorded by dSTORM for the entire synaptic area (D, KS test, p < 0.01). The number of detections of pS270 gephyrin per SSD (E, KS, p = 0.18) and the number of SSDs (F, MW, p = 0.11) were not changed by 4-AP treatment. Gephyrin was probed with mAb7a antibody and Alexa 647 dye. Number of synapses: n = 810 in TTX and n = 727 in 4-AP conditions from three independent experiments; (F) mean ± SEM. Scale bar: 100 nm.


Figure 1B . Flow cytometry analyses of recipient blood 2 months post - transplant . Bi . Transplanted cells detected by GFP fluorescence . Bii . CD11b lebelling shows a large proportion of monocytes expressing GFP . C . Demyelinating lesions in chimeric mice at 3 dpl . Ci . Demyelination was induced by injecting LPC in the spinal cord white matter . Cii - Civ . Co - immunolabeling for GFP , MBP , and a mixture of CD11b / CD68 , showing graft - derived macrophages in the lesion ( identified by lack of MBP staining ) . White lines indicate lesion borders . Scale bar 20 µm . MBP - myelin basic protein ; GFP - green fluorescent protein .

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Figure 1B. Flow cytometry analyses of recipient blood 2 months post-transplant. Bi. Transplanted cells detected by GFP fluorescence. Bii. CD11b lebelling shows a large proportion of monocytes expressing GFP.C. Demyelinating lesions in chimeric mice at 3 dpl. Ci. Demyelination was induced by injecting LPC in the spinal cord white matter. Cii-Civ. Co-immunolabeling for GFP, MBP, and a mixture of CD11b/CD68, showing graft-derived macrophages in the lesion (identified by lack of MBP staining). White lines indicate lesion borders. Scale bar 20 µm. MBP-myelin basic protein; GFP-green fluorescent protein.


Figure 2Transduction of HSCs . B . Lower numbers of GFP + cells , verified using flow cytometry , after transduction with Sema3F vector , * * * p < 0 . 0001 , Student t test , n = 8 independent experiments . Transduction of HSCs . C . Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F - transduced cells only . E . Fold increase in cells that crossed the membrane insert compared to NT . * * * p ‹ 0 . 0001 . One - way ANOVA followed by Bonferroni ' s Multiple Comparison test . n = 1 - 2 normalized replicates from 3 - 5 independent experiments . Mean ± SEM . * * * p < 0 . 0001 . R . = recombinant . Super = supernatant . Anti - Nrp2 = Neuropilin2 function blocking antibody .

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

Figure 2Transduction of HSCs. B. Lower numbers of GFP+ cells, verified using flow cytometry, after transduction with Sema3F vector, ***p<0.0001, Student t test, n=8 independent experiments.Transduction of HSCs. C. Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F-transduced cells only.E. Fold increase in cells that crossed the membrane insert compared to NT. ***p‹0.0001. One-way ANOVA followed by Bonferroni's Multiple Comparison test. n= 1-2 normalized replicates from 3-5 independent experiments. Mean±SEM. ***p<0.0001. R.=recombinant. Super=supernatant. Anti-Nrp2=Neuropilin2 function blocking antibody.


Figure 3Expression of Nrp2 gene by OPCs is maintained in middle - age ( 12 month ( m ) old ) mice . A . Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2 months versus 12 months old PDGFRα − GFP mice ( n = 3 - 4 independent experiments ) . Expression of Nrp2 gene by OPCs is maintained in middle - age ( 12 month ( m ) old ) mice . B . RNA sequencing results for Nrp2 expression by oligodendroglia - enriched preparations isolated from the spinal cords of 2 month versus 12 month old mice . ( n = 3 - 4 independent experiments ) . C - G . Sema 3F expression in demyelinating lesions at 4 dpl ( onset of OPC recruitment ) is decreased in middle - aged ( 12 m old ) and old ( 18m old ) mice compared to young mice ( 2m old ) . C - E . Colabellings for Sema3F and Iba1 ( macrophage marker ) in demyelinating lesions at 4 dpl in 2m ( C ) , 12m ( D ) , and 18m old mice ( E ) . Arrowheads point to double - labelled cells . F . Quantification of Sema3F expressing cells in the lesion . G . Percentage of Iba1 + cells positive for Sema3F . NWM - normal white matter . dpl - days post lesion . n = 4 - 8 mice / group . H . Transwell chamber assay was used to evaluate migration of young versus middle - age OPCs in response to recombinant Sema3F and supernatant from PGK - GFP - T2A - Sema3F transduced cells . OPCs of both ages migrate in response to recombinant Sema3F and PGK - GFP - T2A - Sema3F - transduced cell supernatant . n = 1 - 2 normalized replicates from 2 - 5 independent experiments . Mean ± SEM . One - way ANOVA followed by Bonferroni ' s Multiple Comparison test . * p < 0 . 05 , * * p < 0 . 01 . Scale bar 50 µm .

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 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 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 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

Figure 3Expression of Nrp2 gene by OPCs is maintained in middle-age (12 month (m) old) mice. A. Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2 months versus 12 months old PDGFRα−GFP mice (n=3-4 independent experiments).Expression of Nrp2 gene by OPCs is maintained in middle-age (12 month (m) old) mice. B. RNA sequencing results for Nrp2 expression by oligodendroglia-enriched preparations isolated from the spinal cords of 2 month versus 12 month old mice. (n=3-4 independent experiments).C-G. Sema 3F expression in demyelinating lesions at 4 dpl (onset of OPC recruitment) is decreased in middle-aged (12 m old) and old (18m old) mice compared to young mice (2m old). C-E. Colabellings for Sema3F and Iba1 (macrophage marker) in demyelinating lesions at 4 dpl in 2m (C), 12m (D),and 18m old mice (E). Arrowheads point to double-labelled cells. F. Quantification of Sema3F expressing cells in the lesion. G. Percentage of Iba1+ cells positive for Sema3F. NWM-normal white matter. dpl-days post lesion. n=4-8 mice/group.H. Transwell chamber assay was used to evaluate migration of young versus middle-age OPCs in response to recombinant Sema3F and supernatant from PGK-GFP-T2A-Sema3F transduced cells. OPCs of both ages migrate in response to recombinant Sema3F and PGK-GFP-T2A-Sema3F-transduced cell supernatant. n= 1-2 normalized replicates from 2-5 independent experiments. Mean±SEM. One-way ANOVA followed by Bonferroni's Multiple Comparison test. *p<0.05, ** p<0.01. Scale bar 50 µm.