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 .

O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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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 .

O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A ) 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 ) .

O O O O O B-GENEPROD O O B-TISSUE B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O B-DISEASE I-DISEASE O B-DISEASE O O O O O O B-TISSUE B-DISEASE O O O O O O O O O B-TISSUE O O B-ORGANISM O B-TISSUE O B-TISSUE B-DISEASE O O O O O O O O

A) 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 )

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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 ) .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O B-DISEASE I-DISEASE O B-DISEASE O O

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 .

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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 .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O O O O B-TISSUE O O O O O B-ORGANISM O O O O B-DISEASE O

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 .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O B-DISEASE O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( 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 .

O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(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 .

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(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 .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(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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(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


( 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 .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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 .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(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 .

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(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 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(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.


( 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 .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(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 .

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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 .

O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(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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(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.


( 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 .

O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(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 .

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(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 .

O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(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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(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.


( 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 .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

(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 .

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(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 .

O O O O B-EXP_ASSAY O O O O O B-GENEPROD O 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-SMALL_MOLECULE O O O O O B-GENEPROD O O O O

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 .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

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(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 .

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(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 .

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(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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(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.


( 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 .

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(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 .

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(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 .

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(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 .

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(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.


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

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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

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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

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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

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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

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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

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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 .

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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

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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

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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


TetON - 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 )

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TetON-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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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


hCDC14A , 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

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hCDC14A, 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 μm


Immunoblot 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 .

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Immunoblot 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≤ 0.001


DBN1S142 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 .

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DBN1S142 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≤ 0.001


In 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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In 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


The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate . GAPDH is the loading control .

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The 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 .

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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 .

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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 .

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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 .

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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

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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 .

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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 .

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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.


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

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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

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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

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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

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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

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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


( 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 .

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(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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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.


( 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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(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.


( 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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(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.


( 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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(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.


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


Characterization 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 ) .

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Characterization 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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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).


( 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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(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.


( 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 .

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(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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(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.


B . 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 .

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B. 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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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.


Transduction 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 .

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Transduction 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 .

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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 .

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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.


Expression 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 ) .

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Expression 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 ) .

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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 .

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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 .

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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.


B . Percentage of transplanted ( female ) cells in the blood . n = 18 - 19 mice / group .

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B. 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 .

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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 .

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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 .

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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.


A - 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 .

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A-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 .

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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 .

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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 .

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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 .

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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 + .

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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 .

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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 .

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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.


B - 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 ) .

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B-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 .

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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 .

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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 .

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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.


A - 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 ) .

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A-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 O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O B-CELL_TYPE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O

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.


( 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 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O

(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 .

O O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O O

(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 O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

(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.


( 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O

(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 O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-EXP_ASSAY O

(D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.


( 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 .

O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O O O B-CELL_TYPE O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O B-EXP_ASSAY B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.