( C ) FoxA2 ( green ) Oct4 ( red ) immunostaining of Tcf7l1 null ES on day 4 of differentiation in presence or absence of 3 µM CH . DAPI 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 ) of CXCR4 , Hex Sox17 mRNA by RT - PCR in control wild - type Tcf7l1 null on day 6 of differentiation in presence absence of 3 µM CH . Average SD of three independent experiments .

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(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 ) of FoxA2 mRNA by RT - PCR in FoxA2 - Tet ES lines 48 hr after treatment with indicated dose of DOX . Average SD of two independent experiments from 2 independent lines .

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(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 48 hr after DOX treatment . Left , no DOX control . Middle right , 10ng / ml DOX . DAPI 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 ) of FoxA2 CXCR4 mRNA by RT - PCR on day 3 of differentiation with DOX at indicated dose . Average SD of two experiments from two independent lines .

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(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 ) of Sox17 mRNA by RT - PCR on day 7 of differentiation with addition of 3 µM CH / or 10 ng / ml DOX at indicated time points ( in days ) . Average SD of two independent experiments from two independent lines .

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(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 at day 7 of differentiation in presence or absence of 10 ng / ml DOX presence 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 SD of three independent experiments .

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(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 ) of Tcf7l1 FoxA2 mRNA by RT - PCR at day 2 of differentiation in presence or absence of 3 µM CH or recombinant Wnt3a ( 200 ng / ml plus 500 ng / ml RSpondin1 ( WR ) ) . Average 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 ) of Tcf7l1 FoxA2 mRNA by RT - PCR in Ctnnb1 null at day 1 3 of differentiation in absence or presence of 3 µM CH . Average SD of three independent experiments .

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(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 during first 3 days of differentiation in absence ( - ) or presence ( + ) of 3 µM CH . Results from two independent , clonally - derived Ctnnb1 null ES 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 ) of cMyc mRNA by RT - PCR in wild - type ES at day 1 to 3 of differentiation . Average SD of three independent experiments .

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(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 phospho Ser62 ( pSer62 ) cMyc during first 3 days of differentiation in 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 destabilized form of cMyc ( pThr58 ) during first 3 days of differentiation in absence ( - ) or presence ( + ) of 3 µM CH in wild - type Ctnnb1 null respectively .

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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 ) of Tcf7l1 mRNA by RT - PCR in wild - type at day 2 of differentiation in 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 SD of three independent experiments . * * p = 0 . 0002 .

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(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 ) of Tcf7l1 FoxA2 mRNA by RT - PCR in cMyc null / nMyc heterozygous ( C - / - ; N + / - ) floxed parental ( cMycfl / fl ; nMycfl / fl ( control ) ) at day 3 of differentiation in presence or absence of 3 µM CH . Average 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 cMyc null / nMyc heterozygous ( cMyc - / - ; nMyc + / - ) at day 3 of differentiation in 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 ) of cMyc Tcf7l1 mRNA by RT - PCR in cMycT58A inducible 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 ) of Tcf7l1 mRNA by RT - PCR in icMycT58A clone 1 at day 3 of differentiation in absence of 3 µM CH following cMyc induction with 10 ng / ml DOX . Average 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 Tcf7l1 in icMycT58A clonal 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 differentiated for 2 days in absence or presence of 3 µM CH . qPCR was carried out for regions indicated in Fig . 7a . Average 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 differentiated for 2 days in presence of 3 µM CH plus or minus Myc inhibitor 10058 - F4 . Average 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 ) for Miz1 mRNA by RT - PCR for first three days of differentiation in presence or absence of 3 µM CH . Average 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 differentiated for 2 days in presence of 3 µM CH . qPCR was carried out for regions indicated in Fig . 7a . Average SD for five independent experiments , * * p = 0 . 0015 .

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

(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 hCDC14APD RPE1 were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while basal bodies were marked with C - Nap1 ( red ) . was stained with DAPI . Cilia in magenta box are shown enlarged on 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 hCDC14APD RPE1 were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while basal bodies were marked with C - Nap1 ( red ) . was stained with DAPI of ciliated , as well as of cilia , was quantified from A . Cilia was measured by a semi - automated ImageJ macro as described in methods . Data points are average of three independent experiments with N = 150 cilia for each experiment condition . Error bars represent mean ± SD . * * * * P ≤ 0 . 0001 . High means 80 - 90 % low 40 - 50 % confluency of RPE1

O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-SUBCELLULAR O O 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-SUBCELLULAR O O O O O O O O O O O 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-CELL_LINE

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 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 hCDC14AC278S - GFP under control of TetON promoter were stably integrated into RPE1 . hCDC14A - GFP hCDC14AC278S - GFP localized to C - Nap1 marked basal upon Dox induction . In addition , expression of hCDC14A - GFP but not phosphatase dead hCDC14AC278S - GFP mutant inhibited cilia formation . Ciliated were quantified 48 h after serum starvation with without Dox addition . Three independent experiments ; N = 150 cilia for each experiment 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 hCDC14AC278S - GFP was validated by immunoblot with anti - GFP antibodies . GAPDH was 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 over - expressing hCDC14AC278S have longer cilia than those over - expressing hCDC14A . Three independent experiments , N = 100 cilia for hCDC14A expressing 200 for hCDC14AC278S expressing . 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


dynamics of ciliogenesis was determined by serum starving WT hCDC14APD for different time - points . cilia from hCDC14APD continue to elongate even after 48 h of starvation whereas those from WT reached constant within 12 to 24 h . Three independent experiments ; N = 150 cilia for each experiment condition . Mean ± . * 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 , 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 indicated comparable rates of cilia disassembly in RPE1 WT hCDC14APD . Cilia disassembly rates are shown in Fig . EV1D . Three independent experiments , N = 150 cilia for each experiment condition . Mean ± . * * * * P < 0 . 0001

O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O B-SUBCELLULAR O 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 O O O

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


of cilia of WT RPE1 hCDC14APD RPE1 from I , 0 , 6 24 h after serum addition . Fixed were analyzed for cilia

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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 were serum starved for 48 h prior to fixation immunofluorescence microscopy . hCDC14A - GFP colocalizes with Phalloidin 555 conjugated dye that marks F - actin . boxed was 10 - fold enlarged displayed below corresponding to indicate co - localization of hCDC14A - GFP F - actin . scan inside 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 GFP signal with Arl13B IFT88 ( cilia ) , C - Nap1 / ninein ( proximal end ) , CEP164 ( distal appendage ) ODF2 / ninein ( subdistal appendage ) . Results are fitted in cartoons adjacent to 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 inactive hCDC14AC278S , dephosphorylates serine residue 142 of DBN1 in vivo . RPE1 expressing GFP , hCDC14A - GFP , 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 . 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 of samples from ( A ) . DBN1pS142 / DBN1 was densitometrically measured from Dox treated samples represented in graph next to immunoblot . In this experiment two GAPDH blots were used to normalize because DBN1 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 lines during ciliogenesis compared to WT control . In this experiment we sequentially analyzed DBN1pS142 DBN1 on same blot . specificity of DBN1pS142 antibody was validated by siRNA mediated knock - down of DBN1 in both hCDC14APD GFP / hCDC14A - GFP / hCDC14AC278S - GFP stable 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 expressed for 48 h . DBN1 - GFP was immunoprecipitated from lysates with GFP binder . immunoprecipitated phospho - DBN1 was then incubated with without recombinant hCDC14A in vitro . DBN1pS142 / DBN1 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


absence of DBN1 in targeted clones was further verified by immunoblotting of whole lysate . GAPDH is 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 non - targeted clone 2 . 19 WT control were serum starved for 48 h to induce ciliogenesis . Subsequently , cilia was measured . cilia from DBN1 KO were significantly longer than those from WT or non - targeted controls . Three independent experiments ; N = 150 cilia for each experiment 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 DBN1S142D in RPE1 WT . Three independent experiments , N = 150 cilia for each experiment 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.


phospho - inhibitory ( S142A ) version of DBN1 rescued elongated cilia of DBN1 KO . Three independent experiments , N = 150 cilia for each experiment condition . Mean ± SD . * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 ; ns : not significant . Pairwise comparison was done with unpaired student ' s t - p - value was written in figure . Global comparison was done with one - way ANOVA 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.


overexpressed WT DBN1 was hyper - phosphorylated in as detected by phospho - specific DBN1pS142 antibodies . DBN1pS142 antibodies did not detect 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 confirming successful siRNA depletion of CDK5 indicating hypo - phosphorylation of DBN1S142 in response to CDK5 depletion DBN1pS142 / GAPDH 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 cilia in WT . It restored cilia in hCDC14APD . Three independent experiments ; N = 150 cilia for each experiment 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 decrease can be completely blocked by expression of phospho - mimetic DBN1S142D in hCDC14APD . Three independent experiments ; 150 per experiment 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 ] . transferrin was added for 45 min to . were analyzed after washing . red circle around mother centriole marker CEP164 indicates that was determined for ( B ) . encircled in MERGE is shown 2 - fold enlarged on right side of figure of ( A ) . intensities of transferrin within a 2 μm - radius circle surrounding CEP164 signal were quantified . An ImageJ macro was used to measure as described in methods . Data were calculated from a total of 120 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 showed an increased localization of myosin Va containing vesicles within 1 μm - radius of basal ( red cicle ) . encircled in MERGE is shown 4 - fold enlarged on right side of figure of C . Data was calculated from a total of 180 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 DBN1 KO . Signal was measured 30 min after serum starvation . encircled in MERGE is shown 4 - fold enlarged on right side of 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


of E . Sub - centrosomal ( 1 μm - radius ; red circle in E ) Arp2 was measured by quantifying of Arp2 in WT , hCDC14APD DBN1 KO . Both hCDC14APD DBN1 KO contain significantly higher Arp2 in pericentrosomal ( 1 μm - radius ) compared to WT after 30 min of serum starvation . Data were calculated from a total of 120 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 RPE1 DBN1 KO ( Tet Control ) with DBN1 KO expressing different DBN1 versions ( WT , S142A , S142D ) . Data were calculated from a total of 200 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 characteristics . Standard curves of short , long total CXCL10Simoa assays are shown . For each curve , limit of detection ( LOD ) defined as blank + 3SD , are shown as horizontal lines . LOD was 1 . 7pg / ml for long short CXCL10 assays 0 . 22pg / ml for total CXCL10 . Standard curves were fitted using 4 Parameter Logistic non - linear regression model . Samples reporting a signal below LOD were replaced with 1pg / ml for short 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 ) day 0 ( D0 ) before treatment , at day 3 ( D3 ) , day 14 ( W2 ) day 28 ( W4 ) under treatment 5 after treatment interruption ( W9 ) . ( C ) Plasma from chronic HCVpatients receiving sitagliptin ( n = 3 ) were collected before ( D0 ) weekly during sitagliptin treatment ( W1 W3 ) . Antagonist CXCL103 - 77 ( short CXCL10 , in blue ) , agonist CXCL101 - 77 ( long CXCL10 , in red ) total CXCL10 ( in black ) are shown . Each dot represents a donor bars are at median . Grey areas highlight period under placebo or sitagliptin treatment . Statistical of ( B ) ( C ) was performed using non parametric Friedman ' s , ns : non significant , * * : P0 . 01 , * * * * : P0 . 001 . For ( C ) , additional effect was performed Cohen ' s d values are reported . No statistical was performed in ( D ) due to sample , 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 gephyrin in cryosections . From left to right : rendered of RIM1 / 2 gephyrin clusters showing aligned SSDs ( arrows ) ; gephyrin SSDs segmented by watershed outlined with different colors ; RIM1 / 2 SSDs outlined with different colors ; binary SSDs of gephyrin RIM1 / 2 . Inhibitory synapses were identified by gephyrin clusters in epifluorescence . 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 SSDs of post - synaptic gephyrin , GlyRs , GABAARs , respectively . ( A1 - C1 ) Distances were measured between peaks of paired SSDs in rendered dual - color dSTORM ( yellow , values given in mean ± SD ) . RIM1 / 2 was labelled with Alexa 647 gephyrin ( mAb7a ) with Cy3B in ( A1 ) . RIM1 / 2 was labelled with Cy3B , while GlyRs or GABAARs were labelled with Alexa 647 in ( B1 ) ( C1 ) . ( A2 - C2 ) Trans - synaptic alignment of SSDs of gephyrin , GlyRs , GABAARs 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 . are adjusted to 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 of GlyRs GABAARs at synapses in side view ( A ) or en face view ( B ) . top row of shows pointillist representation of detections , second row rendered bottom row corresponding segmented SSDs . GlyRs were labelled with Alexa 647 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 GABAARs at mixed inhibitory synapses ( arrow ) imaged with conventional 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 nanoscale organization of GABAARs GlyRs at mixed inhibitory synapses . ( A ) Synaptic receptor cluster rendered from detections of both GABAARs ( labelled with Alexa 647 ) GlyRs ( labelled with Cy3B ) . ( B ) Detections of GABAARs ( red dots ) GlyRs ( blue dots ) overlaid with binary synaptic mask ( ) .

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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 GlyR domains . ( F ) Watershed segmentation of sub - synaptic domains ( SSDs ) . Each SSD is shown in different colors . ( G ) Detections of GABAARs ( red dots ) GlyRs ( blue dots ) overlaid with SSDs ( ) .

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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 ) number of receptor SSDs , segmented from combined receptor clusters of GABAARs GlyRs , was decreased after 4 - AP treatment ( MW , p < 0 . 0001 ) . ( B ) This was mostly due to loss of GABAAR SSDs that were segmented from rendered of GABAAR detections only ( MW , * * * * p < 0 . 0001 ) . ( C ) number of GlyR SSDs ( 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 ± .

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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 after 4 - AP treatment , revealed by conventional microscopy ( KS , p < 0 . 001 in B1 p < 0 . 0001 in B2 , p < 0 . 0001 in C ) . Total gephyrin was probed with polyclonal rabbit antibody ( rbGPHN ) , pS270 phosphorylated gephyrin with monoclonal mouse antibody ( m7a ) . Number of synapses : n = 4040 in TTX n = 3818 in 4 - AP conditions from two independent experiments . Scale bar : 2 µm .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-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 O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-EXP_ASSAY B-GENEPROD O O B-ORGANISM O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE 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

(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 entire synaptic ( D , KS , p < 0 . 01 ) . number of detections of pS270 gephyrin per SSD ( E , KS , p = 0 . 18 ) number of SSDs ( F , MW , p = 0 . 11 ) were not changed by 4 - AP treatment . Gephyrin was probed with mAb7a antibody Alexa 647 dye . Number of synapses : n = 810 in TTX n = 727 in 4 - AP conditions from three independent experiments ; ( F ) mean ± . 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 detected by GFP . 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 spinal cord matter . Cii - Civ . Co - immunolabeling for GFP , MBP , a mixture of CD11b / CD68 , showing graft - derived macrophages in lesion ( identified by lack of MBP ) . lines indicate lesion borders . Scale bar 20 µm . MBP - myelin basic ; GFP - green .

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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 + , verified using flow cytometry , after transduction with Sema3F vector , * * * p < 0 . 0001 , Student t , 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 transduced with both vectors Sema3F expression in supernatant of Sema3F - transduced 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 that crossed insert compared to NT . * * * p ‹ 0 . 0001 . One - way ANOVA followed by Bonferroni ' s Multiple Comparison . n = 1 - 2 normalized replicates from 3 - 5 independent experiments . Mean ± . * * * 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 - ( 12 month ( m ) old ) mice . A . Microarray analyses results for Nrp2 expression by OPCs purified using FACS from 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 - ( 12 month ( m ) old ) mice . B . sequencing results for Nrp2 expression by oligodendroglia - enriched preparations isolated from 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 ) old ( 18m old ) mice compared to young mice ( 2m old ) . C - E . Colabellings for Sema3F Iba1 ( macrophage marker ) in demyelinating lesions at 4 dpl in 2m ( C ) , 12m ( D ) , 18m old mice ( E ) . Arrowheads point to double - labelled . F . of Sema3F expressing in lesion . G . of Iba1 + positive for Sema3F . NWM - normal 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 was used to evaluate migration of young versus middle - OPCs in response to recombinant Sema3F supernatant from PGK - GFP - T2A - Sema3F transduced . OPCs of both ages migrate in response to recombinant Sema3F PGK - GFP - T2A - Sema3F - transduced supernatant . n = 1 - 2 normalized replicates from 2 - 5 independent experiments . Mean ± . One - way ANOVA followed by Bonferroni ' s Multiple Comparison . * 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 . of transplanted ( ) in 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 . of transduced ( GFP + ) in blood . Mean ± . * * * 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 + labeled with CD11b , CD19 , CD3 . proportion of GFP + labeled with CD11b is increased in Sema3F mice ( p = 0 . 01 ) , while that of CD19 - labeled is decreased ( p = 0 . 0003 ) . Mean ± . Unpaired Student t - . 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 expressing CD11b , CD19 , CD3 . Red dashed lines indicate expression of these antigens in blood of control mice . n = 9 mice / group . Mean ± .

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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 MBP on spinal cord sections of GFP ( A - C ) Sema3F - GFP chimeras ( D - F ) sacrificed at 7 ( A , D ) , 10 ( B , E ) , 60 ( C , F ) days post lesion ( dpl ) . Absence of MBP labeling in A - B , D - E indicates 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 . of GFP + . Kruskal - Wallis p = 0 . 02 . No significant differences between GFP Sema3F at any of time points ( Dunn ' s post ; Mann - Whitney ) . Decrease in GFP + between 7 60 dpl for both GFP 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 .

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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 T marker CD3 . Arrows indicate CD3 - labeled .

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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 B marker CD45R / B220 . Arrows indicate CD45R / B220 - labeled .

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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 + that are Iba1 + , CD3 + , CD45R / B220 + . absolute majority of GFP + 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 . of Iba1 + , CD3 + , B220 + . For 3 populations , no significant differences in numbers were observed between GFP Sema3F mice , 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 microglial marker P2Y12 . Purple arrows indicate P2Y12 + . Exclusion between two markers is obvious in both groups . Ab . of P2Y12 + . No significant changes are observed between groups or across 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 ) Sema3F ( C , F ) mice . B - C . Co - labeling for PDGFRα , MBP , DAPI . Arrows indicate OPCs in lesion . Bi Ci - insets of dotted squares in B C . D . Higher numbers of PDGFRα + ( OPCs ) in Sema3F mice at 7 dpl ( Mann Whitney : p = 0 . 0031 , n = 6 - 8 mice / group ) . E - F . Co - labeling for Olig2 MBP . E ' - F ' . Co - labeling for Olig2 GFP of lesions shown in E - F . 3D reconstructions . Proximity of OPCs Sema 3F - carrying . E ' i - E ' iii F ' i - F ' iii - insets of E ' - F ' . G . Higher numbers of Olig2 + ( oligodendroglial ) in Sema3F - GFP mice at 7 dpl ( Mann Whitney : 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 + are increased in lesions of Sema3F - GFP mice at 7dpl ( Mann Whitney : p = 0 . 014 , n = 6 - 7 mice / group ) . Arrows indicate Nkx2 . 2 + in close proximity of GFP + .

O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O

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 proliferation marker Ki67 . Arrows indicate double labeled . Q . Numbers of Nkx2 . 2 + Ki67 + are increased at 7 dpl in Sema3F - GFP mice . Mean ± . n = 6 - 7 mice / group . R . proportion of Nkx2 . 2 + co - expressing Ki67 is unchanged . Mean ± . n = 6 - 7 mice / group .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O

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 caspase 3 , marker of apoptosis indicates occasional apoptotic in both groups of mice that are Olig2 - . Arrows indicate caspase 3 + . Y . Numbers of caspase 3 + are same in two groups . Mean ± . n = 6 mice / group .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O

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 lesion border . APC + are scarce in lesions of GFP mice ( A ) . B . APC + in lesion of Sema3F mice . C . of APC + ( Mann Whitney ; p = 0 . 0061 for 7 dpl , p = 0 . 0081 for 10 dpl ; n = 6 - 7 mice / group ) .

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

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 of lesion in GFP ( D ) 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 . of remyelinated axons ( Mann Whitney : p = 0 . 03 , n = 6 mice / group ) . * p < 0 . 05 . H . In Sema3F mice , oligodendroglial ( O ) 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 B-GENEPROD 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 B-SUBCELLULAR 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 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 of immunostaining demonstrate that FKRP - iPSCs express specific pluripotency - associated markers , including NANOG , OCT4 , Tra - 1 - 60 SSEA4 . Data information : Scale bars , 50 μm .

O O O O 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 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 representing ectoderm ( β - III Tubulin , Tuj1 ) , mesoderm ( SMA , smooth muscle actin ) 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 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 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 ) shows precise biallelic correction of FKRP mutation in three independently corrected iPSC clones ( 5D17 , 5D23 3B17 ) , compared with their parental FKRPA455D - iPSCs . Selection cassette excision sites are identified in corrected - iPSC lines .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_TYPE 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 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 B ) Representative of NSCs derived from FKRP - corrected - iPSC lines expressing SOX1 , SOX2 nestin . ( C D ) of of SOX1 + ( C ) SOX2 + ( D ) in culture . efficiency of neural induction is more than 99 % in FKRP - corrected - iPSC ( 5D17 , 5D23 3B17 ) lines . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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

(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 F ) FKRP - corrected NSC lines can be further differentiated to cortical neural progenitor , expressing PAX6 , OTX2 vimentin . ( G - I ) of of PAX6 + ( G ) OTX2 + ( H ) in culture . About 91 - 98 % of derived from FKRP , 5D17 , 5D23 3B17 NSC lines express PAX6 ( G ) . About 93 - 96 % of derived from FKRP , 5D17 , 5D23 3B17 NSC lines express OTX2 ( H ) . Of OTX2 + population , about 60 - 67 % 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 B-GENEPROD O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD 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 B-GENEPROD O O O O O B-CELL_TYPE O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD 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.


( J K ) Glutamatergic projection neurons derived from FKRP corrected ( 5D17 , 5D23 3B17 ) progenitor . vast majority of neurons contain vGlut1 + in their neurites ( labelled by Tuj1 ) . Right panels are enlarged from insets of left panels . Data information : Scale bars , 50 μm .

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

(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.


( A ) Molecular of glycosylated α - dystroglycan ( IIH6 epitope ) in WT - iPSC derived cortical neurons is similar to that in mouse brain ( ~ 120 kDa ) lower than that in mouse muscle ( ~ 156kDa ) . IIH6 reactivity was almost not detected in cortical neurons derived from FKRP - iPSCs . Note that β - Actin antibody does not cross - react with muscle sample .

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

(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.


( B ) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC - derived cortical neurons . Intensities of IIH6 reactivity are normalized to β - Actin expression , indicating mean ± s . d . ( n = 3 biological replicates ; t - ; * * p < 0 . 01 )

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

(B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)


( C ) Representative laminin overlays show that targeted gene correction restores laminin - binding activity in iPSC - derived cortical neurons . Intensities of laminin - binding activity are normalized to β - dystroglycan , indicating mean ± s . d . ( n = 3 biological replicates ; t - ; * p < 0 . 05 ) .

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

(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).


( C ) Dose response curve . H2K 2B4 were treated with serial dilutions of 11 hit compounds for 24 h fixed , stained imaged as described above . average values standard deviations of mean intensities of technical triplicates were calculated a non - linear regression curve was fitted for 3 compounds showing highest increase in α - dystroglycan glycosylation ( IIH6 immunostaining ) . Representative of treated with 5 µM of respective compounds as well as EC50 values are shown . HPFPTzone : ( 5Z ) - 5 - [ ( 3 - ethoxy - 4 - hydroxy - phenyl ) methylene ] - 3 - ( 4 - fluorophenyl ) - 2 - thioxo - thiazolidin - 4 - one . 4BPPNit : 4 - ( 4 - bromophenyl ) - 6 - ethylsulfanyl - 2 - oxo - 3 , 4 - dihydro - 1H - pyridine - 5 - carbonitrile . DPPTzone : ( 5E ) - 5 - [ ( 2 , 5 - dimethyl - 1 - phenyl - pyrrol - 3 - yl ) methylene ] - 3 - phenyl - 2 - thioxo - thiazolidin - 4 - one . Regression curve EC50 values were calculated using GraphPad Prism 7 software . In shown , green represents glycosylated α - dystroglycan blue Hoechst . Scale bar , 50 µm .

(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.


( E ) Flow cytometry confirmed that 4BPPNit treatment ( 20 µM ) significantly increased IIH6 - reactive glycan in mouse H2K 2B4 , compared with untreated . There was no significant difference in of IIH6 - positive . Values indicate mean ± s . e . m . ( n = 6 , triplicates of 2 independent experiments ; t - ; NS , not significant ; * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).


( A ) FKRP - NSCs treated with 4BPPNit showed increased IIH6 reactivity , compared with untreated control . Note that augmented IIH6 reactivity by 4BPPNit is much weaker than that in CRISPR - corrected NSCs . Under same exposure time , signal of IIH6 reactivity is saturated in corrected - NSCs . Data information : Intensities of glycosylation are normalized to GAPDH expression .

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

(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression.


( B ) Representative immunoblots with IIH6 antibody using FKRP - corrected - NSCs treated with 4BPPNit for 24 , 48 72 h , DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH expression . Note that left right panels of immunoblots are not equivalent exposure time .

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

(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time.


( C ) of α - dystroglycan glycosylation ( IIH6 ) , compared with DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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

(C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( D ) Representative immunoblots of laminin - binding using FKRP - corrected - NSCs treated with 4BPPNit DMSO only controls . ( E ) of laminin - binding activities , compared with DMSO only controls . Data information : Intensities of laminin - binding activity are normalized to GAPDH expression . Note that left right panels of immunoblots are not equivalent exposure time . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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

(D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( F ) Representative immunoblots with AF6868 antibody detecting both α - dystroglycan β - dystroglycan in FKRP - corrected - NSCs treated with 4BPPNit DMSO only controls . ( G ) of α - dystroglycan β - dystroglycan expression , compared with DMSO only controls . Data information : Intensities of expression are normalized to GAPDH expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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

(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


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

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

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


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

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

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


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

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


B - D . Mice were analyzed by Kaplan - Meier survival plot ( B ) , of ( C ) , clinical scores ( D ) of WT GPx8 - / - mice with colitis induced by 4 % DSS for 6 days ( n = 10 per group ) . Data information : Data are presented as mean ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ t ‐ ) .

O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-ORGANISM O B-DISEASE 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 O O O O O 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-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( E ) statistical of colon . Data information : Data are presented as mean ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ t ‐ ) .

B-TISSUE O O O O B-SMALL_MOLECULE 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 B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . , 5 - 6 mice were used for each experiment . ( F ) semiquantitative scoring of hematoxylin eosin of colon sections . infiltrated immune at transmural extensions were indicated by arrowhead . Data information : Data are presented as mean ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ t ‐ ) .

B-TISSUE O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colons treated with 4% DSS for 5 days were collected on day 14. , 5-6 mice were used for each experiment. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( G ) Colon sections were stained with markers for macrophages ( F4 / 80 ) anti - GPx8 . Data information : Data are presented as mean ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ t ‐ ) .

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

Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . ( H ) Production of IL - 1β , IL - 6 IL - 18 in colon tissue lysates ( n = 10 per group ) . Data information : Data are presented as mean ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ t ‐ ) .

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

Colons treated with 4% DSS for 5 days were collected on day 14. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . Bray Curtis dissimilarity of fecal microbiota were measured by 16S sequencing ( n = 5 per group ) are presented by a heat map , ( I ) , principal coordinates , ( J ) .

B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-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 O O O O

Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing (n = 5 per group) and are presented by a heat map, (I), and principal coordinates analysis, (J).