Figure 7B ) Paclitaxel treatment did not affect the number of EdU + cells within the HF outer root sheath ( ORS ) following washout . In contrast , palbociclib treated HFs lacked EdU incorporation following drug washout ( adjusted p value = 0 . 0001 [ * * * ] ) . The number of EdU + cells was significantly decreased in the outer root sheath ( ORS ) of HFs dual treated with both paclitaxel and palbociclib ( adjusted p value = 0 . 0021 [ * * ] ) . Ordinary one - way ANOVA with multiple comparisons performed using N of 12 - 13 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard errorC ) HFs treated with paclitaxel showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in the number of cleaved caspase 3 + cells in the ORS following drug washout . Palbociclib - only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3 + ORS cells . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard errorD ) Paclitaxel - only treated HFs showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in the number of pH3 + ORS cells following drug washout . Palbociclib treatment alone had no significant effect on the number of pH3 + ORS cells . There was a trending , yet non - significant , increase in the number of pH3 + ORS cells in HFs dual treated with palbociclib plus paclitaxel . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard errorE ) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout , whereby DNA synthesis has not resumed . ; wo - washout . 50 µm scale ; F ) Representative images of pH3 / caspase 3 staining within the ORS of paclitaxel - only and palbociclib plus paclitaxel treated HFs following drug washout . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 50 µm scaleImmunofluorescence images illustrating how palbociclib blocks the paclitaxel - induced accumulation of cleaved caspase 3 + ( G ) cells in the K15 + bulge and proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale . Immunofluorescence images illustrating how palbociclib blocks the paclitaxel - induced accumulation of pH3 + ( H ) cells in the K15 + bulge and proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

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

Figure 7B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard errorC) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard errorD) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard errorE) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. ; wo - washout. 50 µm scale;F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 50 µm scaleImmunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


Figure 2F . Influence of mutating σ4 on promoter recognition .

Figure 2F. Influence of mutating σ4 on promoter recognition.


Figure 3 B , C . Effects of deleting β ′ ZBD on transcriptional activities of RNAP on different promoters . The fliCp ( B ) was used as template for σ28 - dependent transcription , and the lacUV5p ( C ) was used in σ70 - dependent transcription . RNA products indicated by solid triangles were quantified and the signal obtained from 100 nM wild - type RNAP was normalized to 1 in each test respectively . D . Transcriptional activities of ZBD mutated σ28 - RNAP on fliCp . RNA products indicated by solid triangles were quantified . E . Binding of σ28 - RNAP with fliCp as detected by EMSA ( left ) . The heparin - resistant RNAP - promoter open complex is indicated as " RPo " , and the heparin - sensitive RNAP - promoter closed complex is shown as " RPc " . Percentages of RNAP - promoter open complex ( RPo ) and closed complex ( RPc ) in EMSA performed using σ28 - RNAP and fliCp ( right ) . F . DNase I footprinting of the fliCp complex with σ28 - RNAP . Promoter DNA was labeled on the non - template strand . The promoter region protected by RNAP is indicated by a red box at the bottom panel .

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

Figure 3 B, C. Effects of deleting β′ ZBD on transcriptional activities of RNAP on different promoters. The fliCp (B) was used as template for σ28-dependent transcription, and the lacUV5p (C) was used in σ70-dependent transcription. RNA products indicated by solid triangles were quantified and the signal obtained from 100 nM wild-type RNAP was normalized to 1 in each test respectively.  D. Transcriptional activities of ZBD mutated σ28-RNAP on fliCp. RNA products indicated by solid triangles were quantified.  E. Binding of σ28-RNAP with fliCp as detected by EMSA (left). The heparin-resistant RNAP-promoter open complex is indicated as "RPo", and the heparin-sensitive RNAP-promoter closed complex is shown as "RPc". Percentages of RNAP-promoter open complex (RPo) and closed complex (RPc) in EMSA performed using σ28-RNAP and fliCp (right).  F. DNase I footprinting of the fliCp complex with σ28-RNAP. Promoter DNA was labeled on the non-template strand. The promoter region protected by RNAP is indicated by a red box at the bottom panel.


Figure 4 B , C . Promoter activities of tarp ( B ) and fliCp ( C ) in E . coli wild - type ( Ec - WT ) and its mutants expressing ZBD - mutated RNAP ( Ec - K74A , Ec - K87A and Ec - K74A - K87A ) shown as mean ± SD from three replicates . D . Volcano plot showing log2 - fold change of mRNA abundance as a ratio of Ec - K74A - K87A and Ec - WT strains . Blue dots represent genes with significant difference between these two strains . Red dots represent flagellar genes . Data are shown as mean values of three independent colonies . E . Relative mRNA levels of flagellar genes in Ec - K74A - K87A strain comparing with that in Ec - WT ( normalized to 1 for each gene ) . Diagram for the role of σ28 ( fliA gene ) in flagella regulon hierarchy Clarke & Sperandio , 2005Fitzgerald , 2014 ( ; ) is shown in the left panel . Data are mean ± SEM from three replicates . F . Motilities of Ec - K74A - K87A strains with over - expression of wild - type rpoC gene ( Ec - K74A - K87A - C ) or carrying the control plasmid ( Ec - K74A - K87A - V ) . G . Activities of fliCp in Ec - K74A - K87A - C and Ec - K74A - K87A - V strains . L - arabinose was used at final concentration of 0 . 002 % . Data are mean ± SD from three colonies .

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

Figure 4 B, C. Promoter activities of tarp (B) and fliCp (C) in E. coli wild-type (Ec-WT) and its mutants expressing ZBD-mutated RNAP (Ec-K74A, Ec-K87A and Ec-K74A-K87A) shown as mean ± SD from three replicates.  D. Volcano plot showing log2-fold change of mRNA abundance as a ratio of Ec-K74A-K87A and Ec-WT strains. Blue dots represent genes with significant difference between these two strains. Red dots represent flagellar genes. Data are shown as mean values of three independent colonies.  E. Relative mRNA levels of flagellar genes in Ec-K74A-K87A strain comparing with that in Ec-WT (normalized to 1 for each gene). Diagram for the role of σ28 (fliA gene) in flagella regulon hierarchy Clarke & Sperandio, 2005Fitzgerald, 2014(; ) is shown in the left panel. Data are mean ± SEM from three replicates. F. Motilities of Ec-K74A-K87A strains with over-expression of wild-type rpoC gene (Ec-K74A-K87A-C) or carrying the control plasmid (Ec-K74A-K87A-V). G. Activities of fliCp in Ec-K74A-K87A-C and Ec-K74A-K87A-V strains. L-arabinose was used at final concentration of 0.002%. Data are mean ± SD from three colonies.


Figure 5 B . Activities of chimeric σ28 - R4m in initiating transcription from tarp - 35m , when reconstituted with wild - type ( WT ) or ZBD mutated RNAP core enzyme . Effects of mutating β ′ ZBD on transcriptional activities of σ38 - RNAP on osmYp . Effects of mutating β ′ ZBD on transcriptional activities of σ38 - RNAP on osmYp . Effects of mutating β ′ ZBD on transcriptional activities of σ70 - RNAP on osmYp .

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

Figure 5 B. Activities of chimeric σ28-R4m in initiating transcription from tarp-35m, when reconstituted with wild-type (WT) or ZBD mutated RNAP core enzyme.  Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP on osmYp.  Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP on osmYp.  Effects of mutating β′ ZBD on transcriptional activities of σ70-RNAP on osmYp.


Figure 1 ( A - B ) Immunofluorescence ( IF ) analysis of IκBα in sections from murine small intestine of 2 month - old WT ( A ) and Lgr5 - GFP reporter mice ( B ) . Scale bars , 50μm . IF analysis of P - IκBα in the intestine of 2 month - old WT ( C ) and quantification of P - IκBα positivity Scale bars in C , 25 μmIF analysis of P - IκBα in the intestine of 2 month - old Lgr5 - GFP reporter mice ( D ) , and quantification of P - IκBα positivity inside the Lgr5 - GFP population . A minimum of 30 crypts was counted in 3 Lgr5 - GFP mice . Scale bars in D , 50 μm . ( E ) Distribution of P - IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted . ( F ) Double IF analysis of 2 month - old murine small intestine sections with the indicated antibodies and quantification of P - IκBα positivity inside the EphB2 + population . 70 crypts were counted . Bars represent mean values ± standard error of the mean ( s . e . m ) . Scale bars , 50 μm . ( G ) Double IF analysis of murine small intestine sections with the indicated antibodies . Scale bars , 50 μm . ( H ) Western blot analysis of cytoplasmic ( CYT ) and nuclear ( NUC ) extracts from isolated intestinal crypt cells . ( I ) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti - SUMO2 / 3 antibody . ( J ) IF analysis of developing intestines from E12 . 5 to E17 . 5 with the indicated antibodies . Scale bars , 25 μm . ( K ) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP - sequencing analysis ( n = 4 ) .

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

Figure 1(A-B) Immunofluorescence (IF) analysis of IκBα in sections from murine small intestine of 2 month-old WT (A) and Lgr5-GFP reporter mice (B). Scale bars, 50μm.IF analysis of P-IκBα in the intestine of 2 month-old WT (C) and quantification of P-IκBα positivity Scale bars in C, 25 μmIF analysis of P-IκBα in the intestine of 2 month-old Lgr5-GFP reporter mice (D), and quantification of P-IκBα positivity inside the Lgr5-GFP population. A minimum of 30 crypts was counted in 3 Lgr5-GFP mice. Scale bars in D, 50 μm.(E) Distribution of P-IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted.(F) Double IF analysis of 2 month-old murine small intestine sections with the indicated antibodies and quantification of P-IκBα positivity inside the EphB2+ population. 70 crypts were counted. Bars represent mean values ± standard error of the mean (s.e.m). Scale bars, 50 μm.(G) Double IF analysis of murine small intestine sections with the indicated antibodies. Scale bars, 50 μm.(H) Western blot analysis of cytoplasmic (CYT) and nuclear (NUC) extracts from isolated intestinal crypt cells.(I) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti-SUMO2/3 antibody.(J) IF analysis of developing intestines from E12.5 to E17.5 with the indicated antibodies. Scale bars, 25 μm.(K) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP-sequencing analysis (n=4).


Figure 2 ( A ) Representative images of P4 and P6 mice of the indicated IκBα genotypes . Quantification of the animal weight ( B ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . Quantification of the intestinal length ( C ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . D - G ( D ) Immunohistochemical ( IHC ) , IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines ( D ) , 2 month - old IκBβ ; IκBε KO ( E ) and 2 month - old IκBαNES mice ( F and G ) . A minimum of 50 crypts / villus was counted per genotype ( 3 mice ) . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . Scale bars in D , E , F and G , 50 μm .

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Figure 2(A) Representative images of P4 and P6 mice of the indicated IκBα genotypes.Quantification of the animal weight (B) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.Quantification of the intestinal length (C) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.D-G (D) Immunohistochemical (IHC), IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines (D), 2 month-old IκBβ;IκBε KO (E) and 2 month-old IκBαNES mice (F and G). A minimum of 50 crypts/villus was counted per genotype (3 mice). Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant. Scale bars in D, E, F and G, 50 μm.


Figure 3 ( A ) Representative image of the intestinal populations purified in the cell sorting experiments . ( B ) IF analysis of EphB2 in P6 WT and IκBα KO intestine . Data information : Scale bars , 25 μm . ( C ) P - IκBα IF of EphB2 - high or negative sorted cells . Data information : Scale bars , 25 μm . ( D ) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA - seq . 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq . ( E ) Correlation plot of the indicated differentially expressed gene ( DEG ) sets . Values for DEG in fetal and adult ISC was obtained from Nusse et al , 2012 . ( F ) Table indicating the top 15 up - regulated genes in the IκBα KO EphB2 - high signature including 11 genes in the fetal ISC signature ( in orange ) . ( G ) GSEA of EphB2 - high cells indicating the enrichment of relevant ISC - related pathways . ( H ) Representative image of the Lgr5 + population purified in the cell sorting experiments . ( I ) qPCR analysis of the indicated genes in purified Lgr5 + cells from the different IκBα backgrounds . 4 WT and 1 KO mice were analyzed with at least three technical replicates . Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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Figure 3(A) Representative image of the intestinal populations purified in the cell sorting experiments.(B) IF analysis of EphB2 in P6 WT and IκBα KO intestine. Data information: Scale bars , 25 μm.(C) P-IκBα IF of EphB2-high or negative sorted cells. Data information: Scale bars , 25 μm.(D) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA-seq. 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq.(E) Correlation plot of the indicated differentially expressed gene (DEG) sets. Values for DEG in fetal and adult ISC was obtained from Nusse et al, 2012.(F) Table indicating the top 15 up-regulated genes in the IκBα KO EphB2-high signature including 11 genes in the fetal ISC signature (in orange).(G) GSEA of EphB2-high cells indicating the enrichment of relevant ISC-related pathways.(H) Representative image of the Lgr5+ population purified in the cell sorting experiments.(I) qPCR analysis of the indicated genes in purified Lgr5+ cells from the different IκBα backgrounds. 4 WT and 1 KO mice were analyzed with at least three technical replicates. Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, *p-value<0.05, n.s. no significant.


Figure 4 ( A ) IF analysis of c - Rel and RelA / p65 in P6 WT and IκBα KO intestines . Scale bars in 25 μm . ( B ) Number of peaks from p65 ChIP - sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition ( n = 2 P6 WT and n = 2 P6 IκBα KO intestinal crypt cells ) . Data from individual experiments have been deposited at GSE131187 . IF analysis of active Notch1 ( ICN1 ) ( C ) in the indicated IκBα backgrounds at P6 intestines . Scale bars in 25 μm . IF analysis of β - catenin ( D ) in the indicated IκBα backgrounds at P6 intestines . Scale bars 25 μm . ( E ) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n = 3 P6 animals per genotype were analyzed ( a minimum of 15 crypts was counted per genotype ) . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 . Scale bars 25 μm . ( F ) Table indicating the methylation - related genes ( PRC2 members and KDM6 demethylase ) differentially express between IκBα WT and KO EphB2 - high sorted cells ( RNA - seq ) . ( G ) Western blot analysis of IκBα IP from crypt nuclear extracts . ( I ) Representation of H3K27me3 distribution at embryonic E14 . 5 ( n = 2 WT and n = 2 KO mice ) and postnatal P6 ( n = 2 WT and n = 2 KO mice ) ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of the indicated fetal ISC genes . ( J ) Representation of IκBα ( n = 4 ) and H3K27me3 ( n = 2 ) distribution ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of Litaf .

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

Figure 4(A) IF analysis of c-Rel and RelA/p65 in P6 WT and IκBα KO intestines. Scale bars in 25 μm.(B) Number of peaks from p65 ChIP-sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition (n=2 P6 WT and n=2 P6 IκBα KO intestinal crypt cells). Data from individual experiments have been deposited at GSE131187.IF analysis of active Notch1 (ICN1) (C) in the indicated IκBα backgrounds at P6 intestines. Scale bars in 25 μm.IF analysis of β-catenin (D) in the indicated IκBα backgrounds at P6 intestines. Scale bars 25 μm.(E) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n=3 P6 animals per genotype were analyzed (a minimum of 15 crypts was counted per genotype). Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001. Scale bars 25 μm.(F) Table indicating the methylation-related genes (PRC2 members and KDM6 demethylase) differentially express between IκBα WT and KO EphB2-high sorted cells (RNA-seq).(G) Western blot analysis of IκBα IP from crypt nuclear extracts.(I) Representation of H3K27me3 distribution at embryonic E14.5 (n=2 WT and n=2 KO mice) and postnatal P6 (n=2 WT and n=2 KO mice) (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of the indicated fetal ISC genes.(J) Representation of IκBα (n=4) and H3K27me3 (n=2) distribution (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of Litaf.


Figure 5 ( A ) Representative stereoscopic image of P6 WT and IκBα KO - derived intestinal organoids . Scale bars in A , 100 μm ( B ) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO - derived organoids . Scale bars in B 50 μm . ( C ) qPCR analysis of the indicated ISC genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 . ( D ) Correlation plots of the indicated differentially expressed gene ( DEG ) sets obtained in expression microarray ( 5 technical replicates of a minimum of two organoids per genotype were analyzed ) . Values for DEG in fetal and adult organoids were obtained from Mustata et al , 2013 . ( E ) qPCR analysis of the indicated fetal genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 . ( F ) Western blot analysis of control and IκBα - deleted organoids using CRISPR - Cas9 technology . ( G ) Representative stereoscopic image of WT and IκBα - deleted organoids . Scale bars in G , 50 μm .

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

Figure 5(A) Representative stereoscopic image of P6 WT and IκBα KO-derived intestinal organoids. Scale bars in A, 100 μm(B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids. Scale bars in B 50 μm.(C) qPCR analysis of the indicated ISC genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.(D) Correlation plots of the indicated differentially expressed gene (DEG) sets obtained in expression microarray (5 technical replicates of a minimum of two organoids per genotype were analyzed). Values for DEG in fetal and adult organoids were obtained from Mustata et al, 2013.(E) qPCR analysis of the indicated fetal genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.(F) Western blot analysis of control and IκBα-deleted organoids using CRISPR-Cas9 technology.(G) Representative stereoscopic image of WT and IκBα-deleted organoids. Scale bars in G, 50 μm.


Figure 6A ) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh - RNA for NF - κB elements . Data information Scale bars in A , 100 μm , ( B ) qPCR analysis of the indicated ISC and differentiation genes in the different sh - RNA treated organoids . The canonical NF - κB target gene Cxcl10 is shown as positive control of the experiment . Data information 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant . ( C ) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information Scale bars in 50 μm . ( D - E ) qPCR analysis of the indicated adult and fetal ISC genes ( D ) and elements of the Notch and IFN pathway ( E ) ( differentially expressed in IκBα KO organoids ) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant . ( F ) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor . Data information Scale bars in F , 50 μm . ( G ) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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

Figure 6A) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh-RNA for NF-κB elements. Data information Scale bars in A, 100 μm,(B) qPCR analysis of the indicated ISC and differentiation genes in the different sh-RNA treated organoids. The canonical NF-κB target gene Cxcl10 is shown as positive control of the experiment. Data information 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.(C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information Scale bars in 50 μm.(D-E) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.(F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor. Data information Scale bars in F, 50 μm.(G) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


Figure 7 ( A ) IF analysis of colonic tissue from control and DSS - treated animals including acute damaged ( AD ) areas and recovery ( R ) areas . Data information : Scale bars 100 μmIF analysis of P - IκBα and the indicated markers in the areas of acute damage and recovery ( B ) Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 μm . IF analysis of P - IκBα and colocalization of P - IκBα with the ISC marker Olfm4 in acute damaged areas ( C ) . Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 μm . ( D ) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time - points . 3 technical replicates of organoids from each genotype were analyzed . ( E - F ) Representative images of the colonic tissue from 2 months - old DSS - treated WT and IκBα KO mice are shown . Alcian Blue staining was used to identify the mucus - secreting goblet cells in the colonic glands and Ki67 as proliferation marker . Nuclear counterstain is Fast Red . The table shows number of ulcerations present in the intestines of mice analysed ( 3 WT , 2 IκBα KO ) . Data information : Scale bars in E and F , 100

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

Figure 7(A) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas. Data information: Scale bars 100 μmIF analysis of P-IκBα and the indicated markers in the areas of acute damage and recovery (B) Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.IF analysis of P-IκBα and colocalization of P-IκBα with the ISC marker Olfm4 in acute damaged areas (C). Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.(D) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time-points. 3 technical replicates of organoids from each genotype were analyzed.(E-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO). Data information: Scale bars in E and F, 100 μm


f1a , b , Representative images of GFP - LC3 puncta ( autophagosomes ) in skeletal ( vastus lateralis ) ( a ) and cardiac muscle ( b ) from GFP - LC3 transgenic mice at serial time points after exercise . Scale bar , 20 µm . c , Quantification of data ( mean ± s . e . m . of 10 tissue sections ) in a and b . * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA ) . d , Western blot analysis of LC3 - I / II ( non - lipidated and lipidated forms of MAP1LC3 , respectively ) and p62 levels in indicated tissue from mice at rest ( - ) or after maximal exercise ( + ) . Skeletal and cardiac indicate skeletal and cardiac muscle , respectively . e , Co - immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise . IP , immunoprecipitate ; WB , western blot ; WCL , whole cell lysates .

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f1a, b, Representative images of GFP-LC3 puncta (autophagosomes) in skeletal (vastus lateralis) (a) and cardiac muscle (b) from GFP-LC3 transgenic mice at serial time points after exercise. Scale bar, 20 µm. c, Quantification of data (mean ± s.e.m. of 10 tissue sections) in a and b. **P  0.01, ***P  0.001 (one-way ANOVA).d, Western blot analysis of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in indicated tissue from mice at rest (-) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively.e, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise. IP, immunoprecipitate; WB, western blot; WCL, whole cell lysates.


f2a , Analysis of BCL2 phosphorylation ( detected by anti - BCL2 immunoprecipitation and autoradiography of 32P - labelled cells ) and beclin 1 co - immunoprecipitation with BCL2 in wild - type ( WT ) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle ' s balanced salt solution ( EBSS ) starvation . p - BCL2 , phospho - BCL2 . b , Quantification of GFP - LC3 puncta ( autophagosomes ) in MEFs of indicated genotype in normal growth conditions or starvation conditions . Data represent mean ± s . e . m . for 100 cells per well of triplicate samples per condition . Similar results were observed in three independent experiments . c , Representative images of GFP - LC3 puncta ( autophagosomes ) in skeletal and cardiac muscle of GFP - LC3 wild - type and GFP - LC3BCL2 AAA mice before exercise , after 80 min exercise , or after 75 % of maximal exercise capacity . Scale bar , 20 µm . d , Quantification of data ( mean ± s . d . of 4 mice per experimental group ) in c . e , Western blot analysis of LC3 - I / II and p62 levels in indicated tissue from mice of indicated genotype at rest ( − ) or after maximal exercise ( + ) . Skeletal and cardiac indicate skeletal and cardiac muscle , respectively . f , Co - immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest ( − ) or after 30 min of exercise . WCL , whole cell lysates . NS , not significant . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one - way ANOVA for comparison between groups ; † P 0 . 001 , two - way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes .

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f2a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of 32P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle's balanced salt solution (EBSS) starvation. p-BCL2, phospho-BCL2.b, Quantification of GFP-LC3 puncta (autophagosomes) in MEFs of indicated genotype in normal growth conditions or starvation conditions. Data represent mean ± s.e.m. for 100 cells per well of triplicate samples per condition. Similar results were observed in three independent experiments.c, Representative images of GFP-LC3 puncta (autophagosomes) in skeletal and cardiac muscle of GFP-LC3 wild-type and GFP-LC3BCL2 AAA mice before exercise, after 80 min exercise, or after 75% of maximal exercise capacity. Scale bar, 20 µm. d, Quantification of data (mean ± s.d. of 4 mice per experimental group) in c.e, Western blot analysis of LC3-I/II and p62 levels in indicated tissue from mice of indicated genotype at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively.f, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest (−) or after 30 min of exercise. WCL, whole cell lysates. NS, not significant. *P  0.05, **P  0.01, ***P  0.001, one-way ANOVA for comparison between groups; †P  0.001, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes.


f3a , Maximal treadmill running distance for mice of indicated genotype . Data represent mean ± s . e . m . of 5 mice per group . b , Representative haematoxylin and eosin ( H E ) and periodic acid - Schiff ( PAS ) staining in tibialis anterior muscle sections from mice of indicated genotype . Scale bar , 20 µm . c , d , Plasma glucose ( c ) and insulin ( d ) levels in mice of indicated genotype at rest , after 80 min exercise ( ∼ 900 m ) , or maximal exercise . Data represent combined mean ± s . e . m . for 9 - 11 mice per group from three independent cohorts ; similar results were observed in each cohort . e , Representative images of GLUT4immunofluorescence microscopy staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise . Scale bar , 20 µm . For b and e , similar results were observed in 3 mice per group . f , Western blot analysis of AMPK phosphorylation ( p - AMPK ( Thr172 ) ) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise . g , Maximal treadmill running distance for mice of indicated genotype . Data represent mean ± s . e . m . of 4 - 6 male mice per group . h , Soleus muscle14C - deoxyglucose uptake during treadmill exercise in mice of indicated genotype . Data represent mean ± s . e . m . of 3 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA for comparison between groups ; † P < 0 . 05 , † † P < 0 . 01 , two - way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes . NS , not significant .

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

f3a, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 5 mice per group.b, Representative haematoxylin and eosin (H E) and periodic acid-Schiff (PAS) staining in tibialis anterior muscle sections from mice of indicated genotype. Scale bar, 20 µm.c, d, Plasma glucose (c) and insulin (d) levels in mice of indicated genotype at rest, after 80 min exercise (∼900 m), or maximal exercise. Data represent combined mean ± s.e.m. for 9-11 mice per group from three independent cohorts; similar results were observed in each cohort.e, Representative images of GLUT4immunofluorescence microscopy staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise. Scale bar, 20 µm. For b and e, similar results were observed in 3 mice per group.f, Western blot analysis of AMPK phosphorylation (p-AMPK (Thr172)) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise.g, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 4-6 male mice per group.h, Soleus muscle14C-deoxyglucose uptake during treadmill exercise in mice of indicated genotype. Data represent mean ± s.e.m. of 3 mice per group. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA for comparison between groups; †P<0.05, ††P<0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes. NS, not significant.


f4a , b , Oral glucose tolerance test ( OGTT ) before ( a , week 0 ) and after ( b , week 4 ) 4 weeks of HFD . c , d , OGTT ( c ) and serumleptin and adiponectin levels ( d ) after 8 weeks of daily exercise . For a - d , results represent the mean ± s . e . m . for 4 - 5 mice per group . E , exercise ; NE , no exercise ; RD , regular diet . * P 0 . 05 , * * P 0 . 01 , ( c , one - way ANOVA ; d , Wilcoxon rank test ) . NS , not significant .

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

f4a, b, Oral glucose tolerance test (OGTT) before (a, week 0) and after (b, week 4) 4 weeks of HFD.c, d, OGTT (c) and serumleptin and adiponectin levels (d) after 8 weeks of daily exercise. For a-d, results represent the mean ± s.e.m. for 4-5 mice per group. E, exercise; NE, no exercise; RD, regular diet . *P  0.05, **P  0.01, (c, one-way ANOVA; d, Wilcoxon rank test). NS, not significant.


Figure 1F smFISH of Arglu1 pre - mRNA ( magenta ) and Arglu1 sisRNA ( green ) . Merged dots ( white , arrowheads ) represent Arglu1 pre - mRNA transcript . G Quantification of Arglu1 pre - mRNA and Arglu1 sisRNA copy number from ( F ) . ( n = 50 cells ) Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . I RT - PCR of Arglu1 sisRNA , spliced mRNA levels and actin as control after AMOs treatment .

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

Figure 1F smFISH of Arglu1 pre-mRNA (magenta) and Arglu1 sisRNA (green). Merged dots (white, arrowheads) represent Arglu1 pre-mRNA transcript. G Quantification of Arglu1 pre-mRNA and Arglu1 sisRNA copy number from (F). (n = 50 cells) Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.I RT-PCR of Arglu1 sisRNA, spliced mRNA levels and actin as control after AMOs treatment.


Figure 2G Left : Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells . Right : Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis ( left ) . ( n = 3 biological replicates ) . M Left : RT - PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs . Right : Quantification of relative spliced AXIN1a / AXIN1b expression ratio normalized to actin from RT - PCR analysis ( left ) . ( n = 3 biological replicates ) .

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Figure 2G Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells. Right: Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).M Left: RT-PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs. Right: Quantification of relative spliced AXIN1a/AXIN1b expression ratio normalized to actin from RT-PCR analysis (left). (n = 3 biological replicates).


Figure 3B Left : Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells . Right : Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis ( left ) . ( n = 3 biological replicates ) . D Left : smFISH of Arglu1 sisRNA ( green ) coupled with immunostaining of ARGLU1 protein ( red ) shown in z - stack . Right : Zoom in z - slice on the boxed area showing co - localization ( arrowheads ) of Arglu1 sisRNA with ARGLU1 protein . The intensity plot shows the signal quantification and co - localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow . E Left : smFISH of Arglu1 pre - mRNA ( green ) coupled with ARGLU1 protein ( red ) immunostaining shown in z - stack . Right : Zoom in z - slice on the boxed area showing co - localization ( arrowhead ) of Arglu1 pre - mRNA with ARGLU1 protein . The intensity plot shows the signal quantification and co - localization of Arglu1 pre - mRNA and ARGLU1 protein in the direction of the white arrow . F smFISH of Arglu1 sisRNA ( green ) and Arglu1 pre - mRNA ( magenta ) in 1 day Arglu1 sisRNA KD cells vs control cells . G , H Quantification of Arglu1 sisRNA and Arglu1 pre - mRNA copy number from ( F ) . ( n = 90 cells ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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

Figure 3B Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells. Right: Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).D Left: smFISH of Arglu1 sisRNA (green) coupled with immunostaining of ARGLU1 protein (red) shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowheads) of Arglu1 sisRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow. E Left: smFISH of Arglu1 pre-mRNA (green) coupled with ARGLU1 protein (red) immunostaining shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowhead) of Arglu1 pre-mRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 pre-mRNA and ARGLU1 protein in the direction of the white arrow.F smFISH of Arglu1 sisRNA (green) and Arglu1 pre-mRNA (magenta) in 1 day Arglu1 sisRNA KD cells vs control cells. G, H Quantification of Arglu1 sisRNA and Arglu1 pre-mRNA copy number from (F). (n = 90 cells). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


Figure 4A Immunostaining of MED1 ( magenta ) and SC35 ( red ) with transiently expressed mCherry - ARGLU1 protein ( green ) in MCF - 7 cells . Zoom in of boxed area and the intensity plot shows the co - localization and signal quantification of mCherry - ARGLU1 protein , MED1 and SC35 in the direction of the white arrow . B Confocal images of GFP - SRSF2 ( green , control ) and mCherry - ARGLU1 protein ( red ) at the pre - bleach , bleach , and post - bleach time points in FRAP analysis . The boxed area showing the speckle that was being bleached . C Fluorescence intensities of the nuclear speckle over time in GFP - SRSF2 ( green , control ) and mCherry - ARGLU1 protein ( red ) FRAP analysis . ( n = 3 separate nuclear speckles ) . D Immunostaining of ARGLU1 protein ( green ) and SC35 ( red ) in 1 day Arglu1 sisRNA KD cells vs control cells . Zoom in of boxed area and the intensity plot shows the co - localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow . E Confocal images of 125 nM ARGLU1 - GFP protein ( green ) only and ARGLU1 - GFP protein added with 50 nM IVT RNA ( magenta ) , of either Arglu1 intron 2 with UCE ( Intron 2 WT ) or IVT Arglu1 intron without UCE ( Intron 2 ΔUCE ) . White arrowhead indicates ARGLU1 - GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1 - GFP droplet not incorporated with IVT Intron 2 WT . F Quantification of total ARGLU1 - GFP droplets from ( E ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . G Quantification of average ARGLU1 - GFP droplets size with / without incorporation of IVT intron 2 WT from ( E , ARGLU1 - GFP + Intron 2 WT ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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

Figure 4A Immunostaining of MED1 (magenta) and SC35 (red) with transiently expressed mCherry-ARGLU1 protein (green) in MCF-7 cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of mCherry-ARGLU1 protein, MED1 and SC35 in the direction of the white arrow.B Confocal images of GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) at the pre-bleach, bleach, and post-bleach time points in FRAP analysis. The boxed area showing the speckle that was being bleached. C Fluorescence intensities of the nuclear speckle over time in GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) FRAP analysis. (n = 3 separate nuclear speckles).D Immunostaining of ARGLU1 protein (green) and SC35 (red) in 1 day Arglu1 sisRNA KD cells vs control cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow.E Confocal images of 125 nM ARGLU1-GFP protein (green) only and ARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either Arglu1 intron 2 with UCE (Intron 2 WT) or IVT Arglu1 intron without UCE (Intron 2 ΔUCE). White arrowhead indicates ARGLU1-GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1-GFP droplet not incorporated with IVT Intron 2 WT. F Quantification of total ARGLU1-GFP droplets from (E). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. G Quantification of average ARGLU1-GFP droplets size with/without incorporation of IVT intron 2 WT from (E, ARGLU1-GFP + Intron 2 WT). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.


Figure 6A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells . Samples with rabbit - IgG antibodies were used as control . ARGLU1 antibody # 1 , but not # 2 , worked for IP . F Left : Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control ( sibling control , MTD - GAL4 / CyO ) ovaries . Right : Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis . ( n = 3 biological replicates ) G Left : Immunostaining of dARGLU1 protein ( green ) and VASA ( red ) in dArglu1 sisRNA KD germaria vs control ( sibling control , MTD - GAL4 / TM3 ) germaria . The asterisk ( * ) marks the anterior of the germarium . Right : Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow . Confocal images of 125 nM dARGLU1 - GFP protein ( green ) only and dARGLU1 - GFP protein added with 50 nM IVT RNA ( magenta ) , of either dArglu1 intron 2 until the 3 ' end of sisRNA ( dArglu1 intron 2 ) or IVT dArglu1 intron 2 starting after 3 ' end of sisRNA ( dArglu1 pre - mRNA ) . White arrowheads indicate dARGLU1 - GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1 - GFP droplets not incorporated with IVT dArglu1 intron 2 . J Quantification of total dARGLU1 - GFP droplets from ( I ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum . K Quantification of average dARGLU1 - GFP droplets size with / without incorporation of IVT dArglu1 intron 2 from ( I , dARGLU1 - GFP + dArglu1 intron 2 ) . ( n = 3 biological replicates ) . Cross , mean ; Middle line , median ; box , 25th to 75th percentiles ; whiskers , minimum to maximum .

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

Figure 6A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells. Samples with rabbit-IgG antibodies were used as control. ARGLU1 antibody #1, but not #2, worked for IP.F Left: Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control (sibling control, MTD-GAL4/CyO) ovaries. Right: Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis. (n = 3 biological replicates)G Left: Immunostaining of dARGLU1 protein (green) and VASA (red) in dArglu1 sisRNA KD germaria vs control (sibling control, MTD-GAL4/TM3) germaria. The asterisk (*) marks the anterior of the germarium. Right: Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow.Confocal images of 125 nM dARGLU1-GFP protein (green) only and dARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either dArglu1 intron 2 until the 3' end of sisRNA (dArglu1 intron 2) or IVT dArglu1 intron 2 starting after 3' end of sisRNA (dArglu1 pre-mRNA). White arrowheads indicate dARGLU1-GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1-GFP droplets not incorporated with IVT dArglu1 intron 2. J Quantification of total dARGLU1-GFP droplets from (I). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. K Quantification of average dARGLU1-GFP droplets size with/without incorporation of IVT dArglu1 intron 2 from (I, dARGLU1-GFP + dArglu1 intron 2). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. D


Figure 7A Confocal images of 125 nM d / hARGLU1 - GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT ( magenta ) or dArglu1 intron 2 ( red ) . White arrowheads indicate free IVT RNA dots that are not co - localized with the d / hARGLU1 - GFP droplets . B Quantification of non co - localized RNA dots from ( A , white arrowheads ) . ( n = 16 biological replicates ) . C , D Quantification of total hARGLU1 - GFP droplets and the average droplet size with / without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from ( A ) . ( n = 3 biological replicates ) . E , F Quantification of total dARGLU1 - GFP droplets and the average droplet size with / without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from ( A ) . ( n = 3 biological replicates ) .

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

Figure 7A Confocal images of 125 nM d/hARGLU1-GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT (magenta) or dArglu1 intron 2 (red). White arrowheads indicate free IVT RNA dots that are not co-localized with the d/hARGLU1-GFP droplets. B Quantification of non co-localized RNA dots from (A, white arrowheads). (n = 16 biological replicates). C, D Quantification of total hARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates). E, F Quantification of total dARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).


Figure 1 ( a ) Immunostaining of TCF20 , NESTIN , and merged with DAPI in E13 and E16 embryonic brain sections . TCF20 is widely expressed in the brain . Data information : Scale bar in ( A ) e13 , 50 μm , e16 100 μm ( b ) Costaining with the NSC markers NESTIN , SOX2 , and PAX6 reveals that TCF20 is expressed in NSCs . ( c ) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0 . n = 4 independently repeated tests . ( d ) Western blotting results show the changes in TCF20 protein expression level during brain development . ( e ) Statistical analysis of normalized band intensity in the western blot results . n = 4 samples for each group . ( f ) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution . These electroporated embryos were collected at E16 . ( g ) Statistical analysis of the distributions of GFP - positive cells in different brain regions . The percentage of GFP - positive cells in the CP ( cortical plate ) , IZ ( intermediate zone ) , and VZ / SVZ ( ventricle zone and subventricle zone ) was analyzed . n = 7 brains . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar in ( F ) 50 μm .

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

Figure 1(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm(b) Costaining with the NSC markers NESTIN, SOX2, and PAX6 reveals that TCF20 is expressed in NSCs.(c) Absolute quantitative PCR of TCF20 in different developmental stages from E12 to P0. n=4 independently repeated tests.(d) Western blotting results show the changes in TCF20 protein expression level during brain development. (e) Statistical analysis of normalized band intensity in the western blot results. n= 4 samples for each group.(f) In utero electroporation of TCF20 shRNAs in the E13 cerebral cortex led to abnormal cell distribution. These electroporated embryos were collected at E16. (g) Statistical analysis of the distributions of GFP-positive cells in different brain regions. The percentage of GFP-positive cells in the CP (cortical plate), IZ (intermediate zone), and VZ/SVZ (ventricle zone and subventricle zone) was analyzed. n=7 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.01(**), p<0.001(***). Scale bar in (F) 50 μm.


Figure 2 ( b ) Immunostaining of BrdU . White arrows represent BrdU GFP double - positive cells . Scale bar : 50 μm . ( c ) Statistical analysis of 2 - hour BrdU labeling experiments . n = 6 brains Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( d ) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs . ( e ) Immunostaining with pax6 in E16 electroporated embryo brains . White arrows represent PAX6 , GFP double - positive cells . 1 , 2 are 2x enlarged as the dashed boxes showed . Scale bar : 50 μm . ( f ) Statistical analysis of the percentage of PAX6 + GFP + cells among the total GFP + cells . n = 3 brains for each group . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( g ) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains . White arrows represent GFP , sox2 double positive cells ; Red arrows represent GFP , TBR2 double positive cells . area . Scale bar : 50 μm . ( h ) Statistical analysis of the percentage of SOX2 + GFP + cells among the total GFP + cells . n = 8 brains for each group . ( i ) Statistical analysis of the percentage of TBR2 + GFP + cells among the total GFP + cells . n = 5 brains for each group . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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

Figure 2(b) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm.(c) Statistical analysis of 2-hour BrdU labeling experiments. n=6 brains Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(d) Western blotting analysis indicated an increase in the proliferation marker Sox2 in TCF20 depleted NSCs.(e) Immunostaining with pax6 in E16 electroporated embryo brains. White arrows represent PAX6, GFP double-positive cells. 1,2 are 2x enlarged as the dashed boxes showed. Scale bar: 50 μm. (f) Statistical analysis of the percentage of PAX6+GFP+ cells among the total GFP+ cells. n=3 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(g) Immunostaining with sox2 and pax6 in E16 electroporated embryo brains. White arrows represent GFP, sox2 double positive cells; Red arrows represent GFP, TBR2 double positive cells. area. Scale bar: 50 μm. (h) Statistical analysis of the percentage of SOX2+GFP+ cells among the total GFP+ cells. n=8 brains for each group. (i) Statistical analysis of the percentage of TBR2+GFP+ cells among the total GFP+ cells. n=5 brains for each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


Figure 3 ( a ) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted . White arrows represent SATB2 , GFP double - positive cells . ( b ) Statistical analysis of the SATB2 GFP double - positive cell ratio in GFP positive cells . n = 3 brains . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : 25 μm ( c ) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13 . 5 - E16 . 5 brains . White arrows represent CTIP2 , GFP double - positive cells . White arrow heads represent TBR1 , GFP double - positive cells . ( d , e ) Statistical analysis of CTIP2 , GFP and TBR1 , GFP double - positive cell ratio in GFP positive cells . n = 3 brains . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : ( d ) 50 μm ( f ) A diagram of the BrdU labeling time point in electroporation experiments . ( g ) Immunostaining of BrdU . White arrows represent BrdU GFP double - positive cells . ( h ) Percentage of BrdU and GFP double - positive cells in GFP - positive cells n = 3 brains for each group Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . Scale bar : f ) 25

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

Figure 3(a) SATB2 staining revealed a reduction in SATB2 and GFP colocalization when TCF20 was depleted. White arrows represent SATB2, GFP double-positive cells. (b) Statistical analysis of the SATB2 GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: 25 μm(c) Staining with CTIP2 and TBR1 antibodies shows TCF20 knockdown decreases the colocalization of CTIP2 and TBR1 with GFP in E13.5-E16.5 brains. White arrows represent CTIP2, GFP double-positive cells. White arrow heads represent TBR1, GFP double-positive cells. (d, e) Statistical analysis of CTIP2, GFP and TBR1, GFP double-positive cell ratio in GFP positive cells. n=3 brains. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: (d) 50 μm(f) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: f) 25 μm


Figure 4 ( b ) Western blotting analysis of TCF20 in TCF20 WT , HET , KO mice reveals that TCF20 was deleted . ( c ) Statistical analysis of the normalized band intensity of TCF20 . n = 3 samples for each genotype . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( d ) RT - qPCR analysis of TCF20 in TCF20 WT , HET , and KO mice reveals that TCF20 was deleted . n = 4 samples . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( e ) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out . Scale bar : 50 μm . ( f ) Statistical analysis of the normalized fluorescence intensity of TCF20 . n = 6 samples for each genotype . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( g ) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation . ( h ) Immunostaining of different cortical layer markers TBR1 , CTIP2 , and SATB2 , White dash line represents the positive area . Scale bar : 50 μm . ( i - k ) Statistical analysis of different cells layers in TCF20 WT , HET and KO mice . n = 7 brains of each group . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( l ) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution . Scale bar : 50 μm . ( m ) Statistical analysis of GFP + distribution in different zones . n = 3 brains for each genotype . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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

Figure 4(b) Western blotting analysis of TCF20 in TCF20 WT, HET, KO mice reveals that TCF20 was deleted. (c) Statistical analysis of the normalized band intensity of TCF20. n=3 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(d) RT-qPCR analysis of TCF20 in TCF20 WT, HET, and KO mice reveals that TCF20 was deleted. n=4 samples. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(e) Immunostaining of E16 WT and KO mouse brain slices revealed that TCF20 was successfully knocked out. Scale bar: 50 μm. (f) Statistical analysis of the normalized fluorescence intensity of TCF20. n=6 samples for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(g) Western blotting analysis of neural proliferation and differentiation markers in WT HET and KO mice showed that TCF20 deficits promote cell proliferation and inhibit cell differentiation.(h) Immunostaining of different cortical layer markers TBR1, CTIP2, and SATB2, White dash line represents the positive area. Scale bar: 50 μm. (i-k) Statistical analysis of different cells layers in TCF20 WT, HET and KO mice. n=7 brains of each group. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(l) TCF20 deletion impairs neurogenesis and results in abnormal cell distribution. Scale bar: 50 μm. (m) Statistical analysis of GFP+ distribution in different zones. n=3 brains for each genotype. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


Figure 5 ( a ) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms ( b - d ) ( b ) Total call durations , ( c ) Call durations and ( d ) Call numbers of WT , HET , and KO mouse pups . nWT = 13 mice , nHET = 16 mice , and nKO = 18 mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( e ) A sketch of the mouse trace in the open field test . ( f - h ) ( f ) Statistical analysis of time spent in the center area , ( g ) Statistical analysis of total mouse movement distance in 5 min , ( h ) The ratio of mice in the center area movement distance to the total distance . n for WT = 15 adult mice , n for HET = 12 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( i ) Statistical analysis of the Y maze test . S , O , and N represent the start Arm , old Arm and new Arm . The percent of time that mice stayed in each arm was recorded and analyzed ; n for WT = 11 adult mice , n for HET = 11 adult mice . Whiskers represent the minimum to maximum value ; central lines represent median values ; boxes represent the values between upper quartile and lower quartile . ( j ) Results of the elevated - plus maze . Time that mice spent in open arms ; n for WT = 8 adult mice , and n for HET = 11 adult mice . ( k ) Results of elevated - plus maze . Time that mice spent in closed arms ; n for WT = 8 adult mice , and n for HET = 11 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( l ) Numbers of marbles buried in the marble burying test ; n for WT = 10 adult mice , and n for HET = 10 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) . ( m ) Diagram of the three - chamber social interaction test and mouse movement trace . HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases . ( n ) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1 ; n for WT = 16 adult mice , n for HET = 13 adult mice . ( o ) Comparison between the time that WT and HET mice spent in stranger1 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . ( p ) Time spent in each chamber when one contains familiar mouse stranger1 , as well as new mouse stranger2 . n for WT = 16 adult mice , n for HET = 13 adult mice . ( q ) Comparison between the time of WT and HET mice staying in the stranger2 chamber . n for WT = 16 adult mice , n for HET = 13 adult mice . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) , p < 0 . 001 ( * * * ) .

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

Figure 5(a) Diagram of pup ultrasonic vocalization at the frequency level after Fourier transforms (b-d) (b) Total call durations, (c) Call durations and (d) Call numbers of WT, HET, and KO mouse pups. nWT=13 mice, nHET=16 mice, and nKO=18 mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(e) A sketch of the mouse trace in the open field test. (f-h) (f)Statistical analysis of time spent in the center area, (g) Statistical analysis of total mouse movement distance in 5 min, (h) The ratio of mice in the center area movement distance to the total distance. n for WT=15 adult mice, n for HET=12 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(i) Statistical analysis of the Y maze test. S, O, and N represent the start Arm, old Arm and new Arm. The percent of time that mice stayed in each arm was recorded and analyzed; n for WT=11 adult mice, n for HET=11 adult mice. Whiskers represent the minimum to maximum value; central lines represent median values; boxes represent the values between upper quartile and lower quartile. (j) Results of the elevated-plus maze. Time that mice spent in open arms; n for WT=8 adult mice, and n for HET=11 adult mice. (k) Results of elevated-plus maze. Time that mice spent in closed arms ; n for WT=8 adult mice, and n for HET=11 adult mice.Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(l) Numbers of marbles buried in the marble burying test; n for WT=10 adult mice, and n for HET=10 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).(m) Diagram of the three-chamber social interaction test and mouse movement trace. HET mice did not show preference or stay longer in the stranger1 or stranger2 chambers than the WT mice did in the two test phases. (n) Statistical analysis of the percentage of time spent in each chamber with one empty chamber and another chamber containing stranger1; n for WT=16 adult mice, n for HET=13 adult mice. (o) Comparison between the time that WT and HET mice spent in stranger1 chamber. n for WT=16 adult mice, n for HET=13 adult mice. (p)Time spent in each chamber when one contains familiar mouse stranger1, as well as new mouse stranger2. n for WT=16 adult mice, n for HET=13 adult mice. (q) Comparison between the time of WT and HET mice staying in the stranger2 chamber. n for WT=16 adult mice, n for HET=13 adult mice. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***).


Figure 6 ( c ) Heat map of significantly different genes . ( d ) TDG is significantly decreased after TCF20 deletion . ( e ) Volcano map showing that TDG is significantly decreased . ( f , g ) Electroporation of GFP at E13 . 5 to E16 . 5 in TCF20 KO mice impairs cell distribution . n = 3 brains . Scalebar 25 μm Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . ( h ) Chip - qPCR of TCF20 binding to the TDG promoter . n = 4 biological repeats . Data information : Error bars represent the means ± S . E . M . Two tailed unpaired T - tests were used to analyze the data , n . s . ( no significant difference ) , p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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Figure 6(c) Heat map of significantly different genes.(d) TDG is significantly decreased after TCF20 deletion.(e) Volcano map showing that TDG is significantly decreased.(f, g) Electroporation of GFP at E13.5 to E16.5 in TCF20 KO mice impairs cell distribution. n=3 brains. Scalebar 25 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).(h) Chip-qPCR of TCF20 binding to the TDG promoter. n= 4 biological repeats. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**).