D . Immunoblot of PARP1 cleavage of PeTa after 6 days of indicated treatment . Etoposide serves as positive control for apoptosis , H3 serves as loading control . c . PARP1 , PARP1 .

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D. Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment. Etoposide serves as positive control for apoptosis, H3 serves as loading control. c.PARP1, cleaved PARP1.


E . Caspase - 3 / 7 cleavage activity for PeTa after 6 days of indicated treatment . Etoposide serves as a positive control for apoptosis . n = 3 technical replicates . Data are represented as means ± SD . RLU , relative luminescence units .

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E. Caspase-3/7 cleavage activity for PeTa cell line after 6 days of indicated treatment. Etoposide serves as a positive control for apoptosis. n = 3 technical replicates. Data are represented as means ±SD. RLU, relative luminescence units.


F . Representative FACS readout of tetramethylrhodamine ethyl ester ( TMRE ) stained upon 100 nM GSK - LSD1 or DMSO treatment after 6 days . G . of tetramethylrhodamine ethyl ester ( TMRE ) stained upon 100 nM GSK - LSD1 or DMSO treatment at indicated time points .

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F. Representative FACS readout of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment after 6 days. G. Quantification of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment at indicated time points.


H . Representative FACS readout of Annexin V / DAPI of PeTa after 6 days of 100 nM GSK - LSD1 DMSO treatment . I . of Annexin V / DAPI depicted in Fig 3H . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( Early , Late , Total ; unpaired Student ' s t - ) .

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H. Representative FACS readout of Annexin V/DAPI staining of PeTa cells after 6 days of 100 nM GSK-LSD1 DMSO treatment. I. Quantification of Annexin V/DAPI staining depicted in Fig 3H. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (Early, Late, Total; unpaired Student's t-test).


J . of in vitro TUNEL signal of PeTa after 6 days of 100 nM GSK - LSD1 or vehicle treatment . Representative in Appendix Fig S1B . n = 8 , * * * p = 0 . 001 ( Vehicle vs GSK - LSD1 ; unpaired Student ' s t - ) . ROI , region of interest .

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J. Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK-LSD1 or vehicle treatment. Representative images in Appendix Fig S1B. n = 8, ***p = 0.001 (Vehicle vs GSK-LSD1; unpaired Student's t-test). ROI, region of interest.


K . Top . Representative of immunofluorescent Ki - 67 of slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . of Ki - 67 signal of slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0015 , unpaired Student ' s t - ; D10 vehicle vs D10 GSK - LSD1 p < 0 . 0001 , unpaired Student ' s t - ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 0012 , Mann - Whitney ) . ROI , region of interest .

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K. Top. Representative images of immunofluorescent Ki-67 staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of Ki-67 signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, **p<0.01, ****p<0.0001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0015, unpaired Student's t-test; D10 vehicle vs D10 GSK-LSD1 p<0.0001, unpaired Student's t-test; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.0012, Mann-Whitney test). ROI, region of interest.


L . Top . Representative of immunofluorescent TUNEL of slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . of TUNEL signal of slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * * p < 0 . 001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0009 ; D10 vehicle vs D10 GSK - LSD1 p = 0 . 0001 ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 001 ; unpaired Student ' s t - with Welch ' s correction ) . ROI , region of interest .

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L. Top. Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of TUNEL signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, ***p<0.001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0009; D10 vehicle vs D10 GSK-LSD1 p = 0.0001; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.001; unpaired Student's t-test with Welch's correction). ROI, region of interest.


M . Representative of hematoxylin eosin ( H & E ) of slides from mice treated with GSK - LSD1 or DMSO for 1 day ( D1 , top ) , 10 days ( D10 , middle ) or until experiment endpoint ( bottom ) . Upper right scale bar represents 50 μm , insert scale bar represents 10 μm .

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M. Representative images of hematoxylin and eosin (H&E) staining of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day (D1, top), 10 days (D10, middle) or until experiment endpoint (bottom). Upper right scale bar represents 50 μm, insert scale bar represents 10 μm.


A . Photomicrographs of MCC PeTa after 6 days of 100 nM GSK - LSD1 or DMSO treatment . Scale bar , 100 μm .

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A. Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK-LSD1 or DMSO treatment. Scale bar, 100 μm.


B . Metagene plots showing average CUT & RUN signal for H3K4me1 , H3K27ac , H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start site ; TES transcription end site .

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B. Metagene plots showing the average CUT&RUN signal for H3K4me1, H3K27ac, and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site; TES transcription end site.


C . ATAC - seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start

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C. ATAC-seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site


D . Volcano plot showing - log10 ( adjusted p - value ) log2 fold - change ( log2FC ) for transcripts detected by RNAseq of PeTa treated with 100 nM GSK - LSD1 or DMSO for 6 days . Significantly down - regulated genes ( FDR ≤ 0 . 05 ; log2FC ≤ - 1 ) involved in mitotic cycle ( GO : 0000278 ) are highlighted in blue . E . Upstream promoter motif enrichment of downregulated genes from dataset in Fig 4D . Top - 5 enriched motifs are depicted . TF , transcription factor .

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D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor.


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B . MA - plot of mRNA changes detected by SLAMseq after 1h 6h of 4sU labelling . Significantly up - downregulated genes ( FDR ≤ 0 . 05 ; abs ( log2FC ) ≥ 0 . 5 ) are marked in red blue , respectively . Genes belonging to neuronal differentiation ( GO : 0048666 ) are labelled . FC , fold - change ; CMP , per million .

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B. MA-plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling. Significantly up- and downregulated genes (FDR≤0.05; abs(log2FC)≥0.5) are marked in red and blue, respectively. Genes belonging to neuronal differentiation (GO:0048666) are labelled. FC, fold-change; CMP, counts per million.


H . Gene set enrichment ( GSEA ) of generated Merkel signature on data in Fig 4B . NES , normalized enrichment score ; FDR , false discovery rate .

H. Gene set enrichment analysis (GSEA) of the generated Merkel cell signature on the data in Fig 4B. NES, normalized enrichment score; FDR, false discovery rate.


A . Schematics immunoblot of LSD1 co - immunoprecipitation ( coIP ) from PeTa . IB , Immunoblot ; MS , mass spectrometry .

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A. Schematics and immunoblot of LSD1 co-immunoprecipitation (coIP) from PeTa cells. IB, Immunoblot; MS, mass spectrometry.


B . Volcano plot displaying - binding partners of LSD1 . belonging to LSD1 are marked in red , localization in blue other interaction partners in green . FC , fold - change . C . - interaction ( PPI ) mapping of identified LSD1 binding partners . complexes p - values are displayed in Fig EV 6A .

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B. Volcano plot displaying the protein-protein binding partners of LSD1. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change. C. Protein-protein interaction (PPI) mapping of the identified LSD1 binding partners. Individual complexes and p-values are displayed in Fig EV 6A.


D . Volcano plot depicting - binding partners of LSD1 depleting in PeTa treated with ORY - 1001 versus DMSO . belonging to LSD1 are marked in red , localization in blue other interaction partners in green . FC , fold - change .

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D. Volcano plot depicting the protein-protein binding partners of LSD1 depleting in PeTa cells treated with ORY-1001 versus DMSO. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change.


E . Violin plot depicting HMG20B dependency in MCC compared to cancer types from 23 tissues , ordered according to mean . Red horizontal depicts median . Data obtained from DepMap RNAi screen . Blood , hematopoietic lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system .

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E. Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues, ordered according to mean. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.


G . Volcano plot depicting differentially regulated obtained from Fig 6F . members of LSD1 - CoREST are labeled highlighted in red . significantly upregulated ( - log10 ( p - adjusted ) ≥ 1 ; log2FC ≥ 0 . 75 ) involved in nervous system development ( GO : 0007399 ) are labelled . FC , fold - change .

G. Volcano plot depicting the differentially regulated proteins obtained from Fig 6F. Proteins members of the LSD1-CoREST complex are labeled and highlighted in red. Proteins significantly upregulated (-log10(p-adjusted)≥1; log2FC≥0.75) and involved in nervous system development (GO:0007399) are labelled. FC, fold-change.


H . Left . Immunoblot confirmation of HMG20B downregulation . H3 serves as fraction control α - Tubulin as loading control . Right . Immunoblot . HMG20B are normalized to H3 relative to DMSO control .

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H. Left. Immunoblot confirmation of HMG20B downregulation. H3 serves as nuclear fraction control and α-Tubulin as loading control. Right. Immunoblot quantification. HMG20B protein levels are normalized to H3 and relative to DMSO control.


K . Bar graph of HMG20B rescue experiment in PeTa transduced with either a shRNA targeting HMG20B ( shHMG20B ) or negative control ( shRenilla ) with an overexpression construct expressing either of HMG20B mutant that cannot be targeted by shRNA . survival is depicted at day 8 after transduction normalized to shRenilla . EV , empty vector control , WT , wild - type ; ΔALPHA , alpha - helices deletion ; ΔCC , coiled - coil domain deletion ; ΔHMG , HMG box deletion .

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K. Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B (shHMG20B) or negative control (shRenilla) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA. Cell survival is depicted at day 8 after transduction and normalized to shRenilla. EV, empty vector control, WT, wild-type; ΔALPHA, alpha-helices deletion; ΔCC, coiled-coil domain deletion; ΔHMG, HMG box deletion.


B . RT - qPCR of neuronal genes at different time points conditions depicted in Fig 7A . Data are relative to housekeeping gene HPRT1 normalized to respective DMSO control . n = 4 technical replicates . Bar graphs represent mean ± SD .

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B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD.


C . Immunoblot probing for sustainability of LSD1 activity inhibition at different time points conditions depicted in Fig 7A . α - Tubulin serves as loading control . pSMAD1 / 5 / 9 , phospho - SMAD1 / 5 / 9 . D . Immunoblot probing for LSD1 - CoREST members at different time points conditions depicted in Fig 7A . α - Tubulin serves as loading control .

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C. Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. pSMAD1/5/9, phospho-SMAD1/5/9. D. Immunoblot probing for LSD1-CoREST complex members at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control.


E . Representative FACS readout of EdU / Propidium iodide cycle of PeTa 8 days after drug wash - out . F . of EdU / Propidium iodide depicted in Fig 7E . n = 3 biological replicates . Data are represented as means ± SD . * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0104 ; S phase : DMSO vs GSK - LSD1 p = 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 0062 ; unpaired Student ' s t - ) .

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E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out. F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test).


G . of FACS Annexin V / DAPI of PeTa 8 days after drug wash - out . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( unpaired Student ' s t - ) .

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G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test).


I . Subcutaneous growth of pretreated ( untreated , n = 8 ; D1 , n = 9 ; D3 , n = 9 ; D6 , n = 4 ) Data are represented as means ± . * p < 0 . 05 , * * * p < 0 . 001 ( untreated vs D1 p = 0 . 023 ; untreated vs D3 p = 0 . 023 ; untreated vs D6 p = 0 . 0007 ; Kruskal - Wallis ) .

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I. Subcutaneous tumor growth of pretreated cells (untreated, n = 8; D1, n = 9; D3, n = 9; D6, n = 4) Data are represented as means ±SEM. *p<0.05, ***p<0.001 (untreated vs D1 p = 0.023; untreated vs D3 p = 0.023; untreated vs D6 p = 0.0007; Kruskal-Wallis test).


iPSC - derived HE , EHT HSC - like were sorted , cultured for 1 day analyzed by scRNAseq . ( d ) showing expression of endothelial hematopoietic genes in AEC / Hem cluster which have mapped to HE , EHT HSC - like

iPSC-derived HE, EHT and HSC-like cells were sorted, cultured for 1 day and analyzed by scRNAseq. (d) Heatmap showing expression levels of endothelial and hematopoietic genes in AEC/Hem cluster cells which have mapped to the HE, EHT and HSC-like populations


( b ) Dot plots showing gene expression of glycolytic enzymes detected by scRNAseq on expressed ( of dots ) average level of expression ( color ) .

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(b) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).


( g ) Oxygen consumption rate ( OCR ) was measured in HE EHT ( n = 3 biological replicates with 2 - 3 technical replicates each ) oxidative phosphorylation was assessed by extracellular flux . Bar graphs show relative ± of indicated processes ( from 3 independent experiments ; unpaired t - ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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(g) Oxygen consumption rate (OCR) was measured in HE and EHT cells (n=3 biological replicates with 2-3 technical replicates each) and oxidative phosphorylation was assessed by extracellular flux analysis. Bar graphs show relative levels ± SEM of the indicated processes (from 3 independent experiments; unpaired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( j ) Live imaging of HE HSC - like stained with TMRE ( red ) at day 3 of subculture . Representative merged brightfield / TMRE TMRE are shown . Scale bars , 100 µm . Bar graphs show means of TMRE from replicate wells across experiments ( HE spindle HE round , n = 3 biological replicates with 3 technical replicates each ; HSC - like , n = 2 biological replicates with 3 technical replicates each , Kruskal - Wallis with multiple comparisons ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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(j) Live cell imaging of HE and HSC-like cells stained with TMRE (red) at day 3 of subculture. Representative merged brightfield/TMRE and TMRE images are shown. Scale bars, 100 µm. Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments (HE spindle and HE round, n=3 biological replicates with 3 technical replicates each; HSC-like, n=2 biological replicates with 3 technical replicates each, Kruskal-Wallis test with multiple comparisons). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( k ) Dot plots showing gene expression of TCA cycle enzymes detected by scRNAseq on expressed ( of dots ) average level of expression ( color ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

(k) Dot plots showing gene expression levels of TCA cycle enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( b - e ) FACS - sorted HE were subcultured with or without UK5099 ( 10 µM ) or DCA ( 3 mM ) . Subculture day 3 representative GPA / CD43 plots ( b , d ) subculture day 6 representative CD45 / CD43 plots ( c , e ) are shown Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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(b-e) FACS-sorted HE cells were subcultured with or without UK5099 (10 µM) or DCA (3 mM). Subculture day 3 representative GPA/CD43 plots (b, d) and subculture day 6 representative CD45/CD43 plots (c, e) are shown Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001


( g ) Fold change in expression of HBE1 or HBG1 - 2 transcripts ± normalized to KLF1 in CFUs obtained from HE treated with UK5099 ( 10 µM ) or DCA ( 3 mM ) relative to non - treated ( n = 3 biological replicates , paired t - ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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(g) Fold change in the expression of HBE1 or HBG1-2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non-treated cells (n=3 biological replicates, paired t-test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001


( i - k ) Pregnant mice were injected with UK5099 or DCA at E9 . 5 livers were analyzed at E14 . 5 by flow cytometry . FL , liver . of T B ( i ) LT - HSCs ( j ) as percentages in liver are shown for control ( n = 10 biological replicates ) , UK5099 - treated ( n = 14 biological replicates ) DCA - treated ( n = 16 biological replicates ) conditions ( one - way ANOVA ) . ( k ) of BFU - E to CFU - GM colonies obtained from sorted LT - HSCs are shown CFU , colony forming unit ; BFU , burst forming unit ; E , erythroid ; M , macrophage ; G , granulocyte . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i-k) Pregnant mice were injected with UK5099 or DCA at E9.5 and fetal livers were analyzed at E14.5 by flow cytometry. FL, fetal liver. Levels of T and B cells (i) and LT-HSCs (j) as percentages in fetal liver are shown for control (n=10 biological replicates), UK5099-treated (n=14 biological replicates) and DCA-treated (n=16 biological replicates) conditions (one-way ANOVA test). (k) The ratio of BFU-E to CFU-GM colonies obtained from sorted LT-HSCs are shown CFU, colony forming unit; BFU, burst forming unit; E, erythroid; M, macrophage; G, granulocyte. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001


( l - p ) HE co - cultured with OP9 - DL1 stroma were treated with DCA for 3 days transplanted into irradiated NSG mice . Bone marrow ( BM ) thymi were harvested on week 12 . ( l ) percentages ± of human CD4 + CD8 + double positive thymocytes in huCD45 + from thymus are shown ( Control , n = 6 biological replicates ; DCA , n = 7 biological replicates ; unpaired t ) . percentages ± of human B ( m ) , CLPs ( n ) from BM at week 12 , as well as myeloid from PB at week 8 ( o ) myeloid from BM at week 12 ( p ) in huCD45 + are shown ( Control , n = 6 biological replicates ; DCA , n = 7 biological replicates ; unpaired t ) . PB , peripheral blood . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l-p) HE cells co-cultured with OP9-DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice. Bone marrow (BM) and thymi were harvested on week 12. (l) The percentages ± SEM of human CD4+CD8+ double positive thymocytes in huCD45+ cells from the thymus are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). The percentages ± SEM of human B cells (m), CLPs (n) from the BM at week 12, as well as myeloid cells from PB at week 8 (o) and myeloid cells from BM at week 12 (p) in huCD45+ cells are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). PB, peripheral blood. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001


( d ) Dot plots show expression of indicated hematopoietic transcription factors in clusters 6 7 for HE ctrl , HE + UK5099 HE + DCA conditions , detected by scRNAseq on expressed ( of dots ) average level of expression ( color ) .

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(d) Dot plots show expression levels of the indicated hematopoietic transcription factors in clusters 6 and 7 for HE ctrl, HE + UK5099 and HE + DCA conditions, detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).


( d ) HE were transduced with shScrambled ( shScr ) or shLSD1 with or without UK5099 ( 10 µM ) day after sort day 3 CD43 + / GPA + frequencies ± relative to shScr are presented ( n = 3 biological replicates 1 - 2 technical replicates each , one - way ANOVA ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) HE cells were transduced with shScrambled (shScr) or shLSD1 with or without UK5099 (10 µM) the day after the sort and day 3 CD43+/GPA+ cell frequencies ± SEM relative to shScr are presented (n=3 biological replicates 1-2 technical replicates each, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( f - g ) FACS - sorted HE were subcultured with ACSS2i ( 5 µM ) , DCA ( 3 mM ) , or both ( n = 5 biological replicates , one - way ANOVA ) ( f ) or with C646 ( 10 µM ) , DCA ( 3 mM ) , or both ( n = 3 biological replicates , one - way ANOVA ) ( g ) day 6 CD43 + CD45 + frequencies ± are shown . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f-g) FACS-sorted HE cells were subcultured with ACSS2i (5 µM), DCA (3 mM), or both (n=5 biological replicates, one-way ANOVA test) (f) or with C646 (10 µM), DCA (3 mM), or both (n=3 biological replicates, one-way ANOVA test) (g) and day 6 CD43+CD45+ cell frequencies ± SEM are shown. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( j ) FACS - sorted HE were subcultured with CP ( 5 µM ) , DCA ( 3 mM ) or both day 6 CD43 + CD45 + frequencies ± relative to control are shown ( n = 4 biological replicates , one - way ANOVA ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(j) FACS-sorted HE cells were subcultured with CP (5 µM), DCA (3 mM) or both and day 6 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=4 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( k ) Cholesterol content ± in HE was measured by confocal microscopy at day 2 of DCA ( 3 mM ) treatment by filipin III ( n = 3 biological replicates , paired t ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k) Cholesterol content ± SEM in HE cells was measured by confocal microscopy at day 2 of DCA (3 mM) treatment by filipin III staining (n=3 biological replicates, paired t test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( m ) FACS - sorted HE were subcultured with Ato ( 0 . 5 µM ) , DCA ( 3 mM ) or both day 3 CD43 + CD45 + frequencies ± relative to control are shown ( n = 3 biological replicates for control / DCA , n = 2 biological replicates with 2 technical replicates for Ato / Ato + DCA , one - way ANOVA ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(m) FACS-sorted HE cells were subcultured with Ato (0.5 µM), DCA (3 mM) or both and day 3 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=3 biological replicates for control/DCA, n=2 biological replicates with 2 technical replicates for Ato/Ato+DCA, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


( n ) Dot plots showing gene expression of NOTCH1 , JAG1 HES1 detected by scRNAseq on expressed ( of dots ) average level of expression ( color ) .

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(n) Dot plots showing gene expression levels of NOTCH1, JAG1 and HES1 detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).


( A ) incorporation of 14C - labeled glycerol 3 - phosphate ( G3P ) into various lipids in presence of palmitoyl - CoA ( 10 µM ) in microsomal membranes prepared from HEK293 - AT1 . After incubations at room for 30 min , lipids were extracted separated by TLC before using autoradiography phosphorimaging . Lipid species corresponding to major spots visualized are indicated . Note appearance of phosphatidylcholine ( PC ) cytidine diphosphate - diacylglycerol ( CDP - DAG ) in presence of cytidine trisphosphate ( CTP ) , as well as production of phosphatidylinositol ( PI ) only when both CTP myo - inositol are present . Also , there is a relatively small effect of DAG kinase inhibitor ( DGKi ) on CDP - DAG PI formation in spite of fact that plenty of phosphatidic acid ( PA ) is available from de novo synthesis ( although it should be noted that no ATP was added during these reactions ) . A representative experiment is shown , but these results have been reproduced in other replicates using different treatment combinations . insert shows effect of DGK inhibitor on PI labeling .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O

(A) incorporation of 14C-labeled glycerol 3-phosphate (G3P) into various lipids in the presence of palmitoyl-CoA (10 µM) in microsomal membranes prepared from HEK293-AT1 cells. After incubations at room temperature for 30 min, lipids were extracted and separated by TLC before quantification using autoradiography and phosphorimaging. Lipid species corresponding to the major spots visualized are indicated. Note the appearance of phosphatidylcholine (PC) and cytidine diphosphate-diacylglycerol (CDP-DAG) in the presence of cytidine trisphosphate (CTP), as well as the production of phosphatidylinositol (PI) only when both CTP and myo-inositol are present. Also, there is a relatively small effect of the DAG kinase inhibitor (DGKi) on CDP-DAG and PI formation in spite of the fact that plenty of phosphatidic acid (PA) is available from de novo synthesis (although it should be noted that no ATP was added during these reactions). A representative experiment is shown, but these results have been reproduced in other replicates using different treatment combinations. The insert shows the effect of the DGK inhibitor on PI labeling.


( B ) Effect of lipin - 1γ overexpression on metabolic fate of de novo synthesized PA . Experiments were performed as in panel A , except that membranes were also prepared from expressing a GFP - tagged lipin 1γ , as indicated . Note large amount of TG increased production of PC under these conditions . Results of a representative experiment are shown , these experiments were repeated with identical results .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B) Effect of lipin-1γ overexpression on the metabolic fate of de novo synthesized PA. Experiments were performed as in panel A, except that the membranes were also prepared from cells expressing a GFP-tagged lipin 1γ, as indicated. Note the large amount of TG and increased production of PC under these conditions. Results of a representative experiment are shown, and these experiments were repeated with identical results.


Effect of different fatty - acyl - CoA analogs ( each used in 10 µM ) on metabolic fate of de novo synthesized PA . Note preferential conversion of oleoyl - CoA containing PA into TG very poor incorporation of stearoyl - CoA into TG . Arachidonoyl - CoA is a relatively poor substrate for generation of de novo synthesized PA ; however , this substrate has highest conversion rate into PI , which was calculated as of PI PA mass shown in panel D . , while quantified values ( means ± range ) from two such experiments are shown in ( D ) ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of different fatty-acyl-CoA analogs (each used in 10 µM) on the metabolic fate of de novo synthesized PA. Note the preferential conversion of oleoyl-CoA containing PA into TG and the very poor incorporation of stearoyl-CoA into TG. Arachidonoyl-CoA is a relatively poor substrate for the generation of de novo synthesized PA; however, this substrate has the highest conversion rate into PI, which was calculated as the ratio of the PI and PA mass shown in panel D. , while quantified values (means ± range) from two such experiments are shown in (D) and (E).


( E F ) RT - PCR western blot showing effect of RNAi - mediated B52 depletion in S2R + ( E ) in larval wing discs ( F ) .

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(E and F) RT-PCR and western blot showing the effect of RNAi-mediated B52 depletion in S2R+ cells (E) and in larval wing discs (F).


( D ) of Posterior / Total wing in flies of genotypes illustrated in panel C . UAS - GFP was used as a control . Each point represents a single wing , bars represent mean with standard deviation . * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ( unpaired two - t - ) . Flies were grown at 18 ° C .

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

(D) Quantification of Posterior/Total wing area in male flies of the genotypes illustrated in panel C. UAS-GFP was used as a control. Each point represents a single wing, bars represent mean with standard deviation. *** p-value<0.001, **** p-value<0.0001 (unpaired two-tailed t-tests). Flies were grown at 18°C.


( C ) of Yki reporter genes expression upon overexpression of Yki isoforms in wing posterior domain ( hh - Gal4 driver ) . For each target gene , relative expression between posterior anterior domain in wing discs was quantified by immunostaining using anti - ßgal antibody ( for ex - lacZ diap1 - lacZ ) or direct visualization of GFP ( ban - sensor , in this case posterior domain was visualized by immunostaining against V5 - tag present in UAS - Yki transgenes ) . In scatter dot plot , each symbol ( circle , square triangle ) represents a single wing disc , bars represent mean with . * * p - value < 0 . 01 ; * * * * p - value < 0 . 0001 ( unpaired two - t - ) .

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

(C) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain (hh-Gal4 driver). For each target gene, relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti-ßgal antibody (for ex-lacZ and diap1-lacZ) or direct visualization of GFP (ban-sensor, in this case posterior domain was visualized by immunostaining against V5-tag present in UAS-Yki transgenes). In the scatter dot plot, each symbol (circle, square and triangle) represents a single wing disc, bars represent mean with SEM. ** p-value<0.01; **** p-value<0.0001 (unpaired two-tailed t-tests).


( C ) Comparison of Yki isoforms binding capabilities by dual - luciferase co - IP . Graphs show normalized co - IP ( NcoIP ) results for 13 tested against two Yki isoforms negative control ( Flag - Firefly ) .

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(C) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly).


( D ) Interaction between Sd Tgi in absence or presence of Yki isoforms . ( E ) Interaction between Sd Wbp2 in absence or presence of Yki isoforms . ( F ) Interaction between Tgi Wbp2 in absence or presence of Yki isoforms . In C , D , E F , bars represent mean error bars represent SD , n = 4 . p - values : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( unpaired two - t - ) .

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(D) Interaction between Sd and Tgi in absence or presence of Yki isoforms. (E) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms. (F) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms. In C, D, E and F, bars represent mean and error bars represent SD, n=4. p-values: *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed t-tests).


( G ) Luciferase using 3xSd - Luciferase reporter in S2 , transfected with single or combination of Yki isoforms . Error bars represent SD , n = 6 . * p - value < 0 . 05 ; * * * * p - value < 0 . 0001 ( unpaired two - t - ) .

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(G) Luciferase assay using 3xSd-Luciferase reporter in S2 cells, transfected with single or combination of Yki isoforms. Error bars represent SD, n=6. * p-value<0.05; **** p-value<0.0001 (unpaired two-tailed t-tests).


( H ) Co - overexpression of Yki isoforms using GMR - Gal4 driver provides evidence of competition between Yki isoforms .

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(H) Co-overexpression of Yki isoforms using GMR-Gal4 driver provides evidence of competition between Yki isoforms.


( C ) Comparison of posterior / total of wings upon depletion of B52 in a yki + ( wild type ) or yki2 - only background . Each point corresponds to a single wing , bars represent mean with SD ( unpaired two - t - : * * * * p - value < 0 . 0001 ) .

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(C) Comparison of posterior/total area ratio of females wings upon depletion of B52 in a yki+ (wild type) or yki2-only background. Each point corresponds to a single wing, bars represent mean with SD (unpaired two-tailed t-tests: **** p-value <0.0001).


( E F ) of sibling clones in wing pouch . In ( D ) , sibling wild - type clones are labelled with 2xGFP 2xRFP . In ( E ) , unlabelled clones are homozygous for yki2 - only - A allele whereas sibling clones ( RFP / RFP ) are wild type . In both graphs , clones pairs are sorted according to RFP / RFP clones .

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(E and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.


( C ) Distribution of wing wing asymmetry among flies analyzed in panel B . Each circle represents of a single wing . Each wing was measured in duplicate . difference between Right ( orange ) Left ( blue ) sides of a single fly is schematized by a black .

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

(C) Distribution of wing size and wing asymmetry among the flies analyzed in panel B. Each circle represents the area of a single wing. Each wing was measured in duplicate. The difference between Right (orange) and Left (blue) sides of a single fly is schematized by a black line.


C . Representative western blot showing changes in various ER stress markers O - GlcNAcylation - related expression in THP - 1 treated with Stx2a ( 10 ng / mL ) for 3 h in presence or absence of O - linked N - acetylglucosamine transferase ( OGT ) inhibitor OSMI - 1 ( 10 µM final ) or serine / threonine - kinase / endoribonuclease inositol - requiring enzyme 1 a ( IRE1a ) inhibitor MKC - 3946 ( 10 µM final ) . D . of band intensities in ( C ) . Data are presented as mean ± S . E . M . ( n = 3 biological replicates ) normalized against β - actin , which was used as a loading control . Data information : Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative western blot showing changes in various ER stress markers and O-GlcNAcylation-related protein expression in THP-1 cells treated with Stx2a (10 ng/mL) for 3 h in the presence or absence of O-linked N-acetylglucosamine transferase (OGT) inhibitor OSMI-1 (10 µM final) or serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 a (IRE1a) inhibitor MKC-3946 (10 µM final). D. Quantification of the band intensities in (C). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


E . Representative western blot of O - GlcNAcylation pro - caspase - 3 cleavage in THP - 1 treated with Stx2a ( 10 ng / mL ) in presence or absence of OGT inhibitor OSMI - 1 ( 10 µM final ) . F . of band intensities for O - GlcNAcylation ( RL - 2 ) in ( E ) . Data are presented as mean ± S . E . M . ( n = 3 biological replicates ) normalized against β - actin , which was used as a loading control . effects of Stx2a - mediated induction for O - GlcNAc at each time - point were compared to 0 h ( left panel ) , OSMI - 1 treatment was compared with that of vehicle ( DMSO ) control at each time - point ( right panel ) . Data information : Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative western blot of O-GlcNAcylation and pro-caspase-3 cleavage in THP-1 cells treated with Stx2a (10 ng/mL) in the presence or absence of OGT inhibitor OSMI-1 (10 µM final). F. Quantification of the band intensities for O-GlcNAcylation (RL-2) in (E). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. The effects of Stx2a-mediated induction for O-GlcNAc levels at each time-point were compared to 0 h (left panel), and OSMI-1 treatment was compared with that of the vehicle (DMSO) control at each time-point (right panel). Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


B . ELISAs were used to analyze inhibitory effect of OSMI - 1 ( 10 µM , final ) on cytokine chemokine production from THP - 1 exposed to Stx2a ( 10 ng / mL ) for 9 h ( n = 3 biological replicates ) . Data information : For graphs error bar represents mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . effects of OSMI - 1 were compared with those of vehicle ( DMSO ) control at each time - point .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

B. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on cytokine and chemokine production from THP-1 cells exposed to Stx2a (10 ng/mL) for 9 h (n = 3 biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.


C . Real - time qRT - PCR analyses of fold changes in transcript for each cytokine / chemokine gene in D - THP - 1 following Stx2a ( 10 ng / mL ) intoxication for various time - points in presence or absence of OSMI - 1 ( 10 µM , final ) . Data are normalized using GAPDH as a loading control ( n = 3 , biological replicates ) . Data information : For graphs error bar represents mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . effects of OSMI - 1 were compared with those of vehicle ( DMSO ) control at each time - point .

O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

C. Real-time qRT-PCR analyses of fold changes in transcript levels for each cytokine/chemokine gene in D-THP-1 cells following Stx2a (10 ng/mL) intoxication for various time-points in the presence or absence of OSMI-1 (10 µM, final). Data are normalized using GAPDH as a loading control (n = 3, biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.


A . Representative western blot showing changes of overall O - GlcNAcylation ( upper panel ) or phosphorylation status of each target ( lower panels ) in THP - 1 treated with Stx2a ( 10 ng / mL ) for 9 h in presence or absence of OSMI - 1 ( 10 µM , final ) . B . of relative band intensities for O - GlcNAcylation ( RL - 2 ) or each of phosphorylated - total - bands in ( A ) . Data are normalized against β - actin , which was used as a loading control ( n = 3 biological replicates ) . effects of Stx2a in vehicle controls were compared with lysates prepared from maintained in absence of Stx2a , OSMI - 1 treatment was compared with that of vehicle ( DMSO ) controls in presence of Stx2a . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative western blot showing changes of overall O-GlcNAcylation (upper panel) or phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of OSMI-1 (10 µM, final). B. Quantification of the relative band intensities for O-GlcNAcylation (RL-2) or each of the phosphorylated- and total-protein bands in (A). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with lysates prepared from cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C . Representative western blot showing changes in OGT expression ( top panel ) , overall O - GlcNAcylation ( second panel ) , or phosphorylation status of each target ( lower panels ) in THP - 1 treated with Stx2a ( 10 ng / mL ) for 9 h in presence or absence of OGT knockdown . D . of relative band intensities between each of phosphorylated - total - bands in ( C ) . Data are normalized against β - actin , which was used as a loading control ( n = 3 biological replicates ) . effects of Stx2a in siScr controls were compared with those of lysates from maintained in absence of Stx2a , OGT knockdown was compared with that of siScr controls in presence of Stx2a . ( n . s . = not significant ) . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

C. Representative western blot showing changes in OGT expression (top panel), overall O-GlcNAcylation (second panel), or phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of OGT knockdown. D. Quantification of the relative band intensities between each of phosphorylated- and total-protein bands in (C). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in siScr controls were compared with those of lysates from cells maintained in the absence of Stx2a, and OGT knockdown was compared with that of siScr controls in the presence of Stx2a. (n.s. = not significant). Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


G . Representative western blot showing changes of overall O - GlcNAcylation ( upper panel ) , phosphorylation status of each target ( middle panels ) or pro - caspase - 3 cleavage ( lower panel ) in THP - 1 treated with Stx2a ( 10 ng / mL ) for 9 h in presence or absence of overexpression of Akt ( wild - type or T305A / T312A ) . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

G. Representative western blot showing changes of overall O-GlcNAcylation (upper panel), phosphorylation status of each target protein (middle panels) or pro-caspase-3 cleavage (lower panel) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of overexpression of Akt (wild-type or T305A/T312A). Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


A . Representative western blot showing changes of O - GlcNAcylation in human renal proximal tubule epithelial ( HRPTEpi ) ( originating from or adult kidney ) treated with different concentrations ( 0 - 10 ng / mL ) of Stx2a for 24 h .

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

A. Representative western blot showing the changes of O-GlcNAcylation in primary human renal proximal tubule epithelial (HRPTEpi) cells (originating from fetal or adult kidney) treated with different concentrations (0-10 ng/mL) of Stx2a for 24 h.


B . LDH cytotoxicity following Stx2a ( 10 ng / mL ) treatment of HRPTEpi ( adult ) for 48 h in presence or absence OSMI - 1 ( 10 µM , final ) ( n = 3 biological replicates ) . effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi maintained in absence of Stx2a , OSMI - 1 treatment was compared with that of vehicle ( DMSO ) controls in presence of Stx2a . Data information : For graphs error bar represents mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. LDH cytotoxicity assay following Stx2a (10 ng/mL) treatment of HRPTEpi (adult) cells for 48 h in the presence or absence OSMI-1 (10 µM, final) (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C . ELISAs were used to analyze inhibitory effect of OSMI - 1 on cytokine / chemokine production from HRPTEpi ( adult ) exposed to Stx2a ( 10 ng / mL ) for 48 h ( n = 3 biological replicates ) . effects of OSMI - 1 were compared with those of vehicle ( DMSO ) control . Data information : For graphs , error bar represents mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on cytokine/chemokine production from HRPTEpi cells (adult) exposed to Stx2a (10 ng/mL) for 48 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) control. Data information: For graphs , error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C . ELISAs were used to analyze inhibitory effect of OSMI - 1 on IL - 8 CCL - 2 production from 3D cultures of HRPTEpi exposed to Stx2a ( 10 ng / mL ) for 72 h ( n = 3 biological replicates ) . effects of OSMI - 1 were compared with those of vehicle ( DMSO ) controls . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of HRPTEpi cells exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


F . ELISAs were used to analyze inhibitory effect of OSMI - 1 on IL - 8 CCL - 2 production from 3D cultures of human mini - kidneys exposed to Stx2a ( 10 ng / mL ) for 72 h ( n = 3 biological replicates ) . effects of OSMI - 1 were compared with those of vehicle ( DMSO ) controls . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of human mini-kidneys exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


G , H . Human apoptosis antibody array of multiple using pooled lysate from 3D - human mini - kidney spheroids following Stx2a ( 10 ng / mL ) treatment for 72 h in presence or absence OSMI - 1 ( 10 µM , final ) . ( G ) Antibody spots representing signal differences are indicated in red boxes . ( H ) graph shows average of relative spot intensities for each compared to those measured in control in absence of Stx2a exposure ( n = 2 biological replicates ) . Dashed represents reference point of fold change . Raw values of intensities are provided in Source Data . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H. Human apoptosis antibody array analysis of multiple proteins using pooled lysate from 3D-human mini-kidney spheroids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (G) Antibody spots representing signal differences are indicated in red boxes. (H) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


C , D . ELISAs were used to analyze inhibitory effect of OSMI - 1 ( 10 µM , final ) on Kidney injury molecule - 1 ( KIM - 1 ) secretion ( C ) IL - 8 CCL - 2 production ( D ) in culture supernatants of iPSC - derived human kidney organoids exposed to Stx2a ( 10 ng / mL ) for 72 h ( n = 3 biological replicates ) . effects of OSMI - 1 were compared with those of vehicle ( DMSO ) controls . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on Kidney injury molecule-1 (KIM-1) secretion (C) and IL-8 and CCL-2 production (D) in culture supernatants of iPSC-derived human kidney organoids exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


E , F . Human apoptosis antibody array of multiple using pooled lysates from iPSC - derived human kidney organoids following Stx2a ( 10 ng / mL ) treatment for 72 h in presence or absence OSMI - 1 ( 10 µM , final ) . ( E ) Antibody spots representing signal differences are indicated in red boxes . ( F ) graph shows average of relative spot intensities for each compared to those measured in control in absence of Stx2a exposure ( n = 2 biological replicates ) . Dashed represents reference point of fold change . Data information : Error bars for bar graphs are presented as mean ± S . E . M . Statistical was performed using two - Student ' s t - . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F. Human apoptosis antibody array analysis of multiple proteins using pooled lysates from iPSC-derived human kidney organoids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (E) Antibody spots representing signal differences are indicated in red boxes. (F) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.


B . Survival rate of mice challenged once with Stx2a ( 132 . 5 ng / kg ) injected daily with two doses ( 300 or 1000 µg / mouse ) of OSMI - 1 ( n = 10 , 5 per dose ) or vehicle ( n = 10 ) . effect of OSMI - 1 ( 1000 µg / mouse ) was compared with that of vehicle control ( a log - rank with GraphPad Prism software ) .

O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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. Survival rate of mice challenged once with Stx2a (132.5 ng/kg) and injected daily with two doses (300 or 1000 µg/mouse) of OSMI-1 (n = 10, 5 per dose) or vehicle (n = 10). The effect of OSMI-1 (1000 µg/mouse) was compared with that of the vehicle control (a log-rank test with GraphPad Prism software).


C . Chemical hematological analyses of blood pooled from three mice at day 3 after Stx2a challenge . Data are presented as mean ± S . E . M . ( n = 3 biological replicates from nine mice , two - Student ' s t - ) . effects of OSMI - 1 ( 1000 µg / mouse ) were compared with those of vehicle control . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Chemical and hematological analyses of blood pooled from three mice at day 3 after Stx2a challenge. Data are presented as mean ± S.E.M. (n = 3 biological replicates from nine mice, two-tailed Student's t-test). The effects of OSMI-1 (1000 µg/mouse) were compared with those of the vehicle control. *P < 0.05; **P < 0.01; and ***P < 0.001


( C ) Brucella replication in BMMs expressing either GFP or GFP - BspF infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) , measured as number of per at 24 h pi . Data are means ± SD of n = 3 independent experiments . Grey dots represent values from analyzed ( n > 300 ) ; black dots indicate means of experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons ) compared to controls .

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

(C) Brucella replication in BMMs expressing either GFP or GFP-BspF and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to controls.


( A ) of ss - eGFP - FKBPF36M trafficking in HeLa ( M ) - C1 transfected for 24 h with pmCherry ( mCherry ) or pmCherry - BspF ( mCherry - BspF ) . Rapamycin was added to initiate secretory traffic of ss - eGFP - FKBPF36M its colocalization with calnexin ( ER ) , ERGIC - 53 ( ERGIC ) , GM130 ( Golgi ) , p230 ( TGN ) , or secretory vesicles ( SV ) was scored over a 60 min time course . Data are means ± SD from n = 3 independent experiments . Asterisks indicate statistically significant differences between mCherry - mCherry - BspF - expressing as determined by a two - way ANOVA with Sidak ' s multiple comparisons ( P < 0 . 05 ) .

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

(A) Quantification of ss-eGFP-FKBPF36M trafficking in HeLa(M)-C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry-BspF (mCherry-BspF). Rapamycin was added to initiate secretory traffic of ss-eGFP-FKBPF36M and its colocalization with calnexin (ER), ERGIC-53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) was scored over a 60 min time course. Data are means ± SD from n=3 independent experiments. Asterisks indicate statistically significant differences between mCherry- and mCherry-BspF-expressing cells as determined by a two-way ANOVA with Sidak's multiple comparisons test (P<0.05).


( F ) Representative confocal of HeLa co - transfected for 24 h to produce mCherry - BspF either GFP - Rab6a , GFP - Rab8a or Arf6 - GFP treated with Cytochalasin D ( 200 nM ) for 30 min prior to fixation . Scale bars : 10 2 µm ( insets ) . ( G ) of localization of GFP - Rab6a , GFP - Rab8a Arf6 - GFP on mCherry - BspF - labelled tubules in transfected HeLa . Data are means ± SD from n = 3 independent experiments , in which at least 300 per experiment were analyzed .

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

(F) Representative confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce mCherry-BspF and either GFP-Rab6a, GFP-Rab8a or Arf6-GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). (G) Quantification of localization of GFP-Rab6a, GFP-Rab8a and Arf6-GFP on mCherry-BspF-labelled tubules in transfected HeLa cells. Data are means ± SD from n=3 independent experiments, in which at least 300 individual cells per experiment were analyzed.


( A ) Representative confocal of HeLa transfected for 24 h to produce either mCherry or mCherry - BspF ( grayscale panels ) , incubated on ice with AlexaFluor ™ 488 - Cholera Toxin subunit B ( CTxB ; green ) shifted to 37 ˚ C for 20 min to allow for CTxB retrograde transport to Golgi apparatus ( stained using an anti - GM130 antibody ; purple ) . CTxB accumulation within Golgi structures appears in overlays . Scale bars : 10 2 µm ( insets ) . ( B ) of CTxB transport to Golgi apparatus in HeLa producing either mCherry , mCherry - BspF or HA - BspF over a 30 min time course , expressed as percentages of in which CTxB colocalized with GM130 Golgi marker , as in ( A ) . Data are means ± SD from n = 3 independent experiments , in which 100 were analyzed per experiment . Asterisks indicate statistically significant differences compared to mCherry - producing as determined by a two - way ANOVA with Tukey ' s multiple comparisons ( P < 0 . 05 ) . (

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

(A) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry-BspF (grayscale panels), incubated on ice with AlexaFluor™488-Cholera Toxin subunit B (CTxB; green) and shifted to 37˚C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti-GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). (B) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry-BspF or HA-BspF over a 30 min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared to mCherry-producing cells as determined by a two-way ANOVA with Tukey's multiple comparisons test (P<0.05). (


( D ) of CTxB transport to Golgi apparatus in BMMs following siRNA - mediated depletion of either Arf6 ( siArf6 ) , Rab8a ( siRab8a ) or Rab6a / a ' ( siRab6a / a ' ) after AlexaFluor ™ 488 - CTxB binding on ice followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 were analyzed per experiment . Asterisks indicate a statistically significant difference compared to siNT control as determined by a one - way ANOVA with Tukey ' s multiple comparisons ( P < 0 . 05 ) . ( E ) of CTxB transport to Golgi apparatus in BMMs that were either mock - infected or infected with wild type ( 2308 ) , ΔbspF , complemented ∆ bspF ( ΔbspF : : bspF ) , ∆ bspB or complemented ∆ bspB ( ΔbspB : : bspB ) for 24 h , incubated for 30 min with AlexaFluor ™ 488 - CTxB on ice for binding followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 were analyzed per experiment . Asterisks indicate a statistically significant difference compared to mock - infected as determined by a one - way ANOVA with Tukey ' s multiple comparisons ( P < 0 . 05 ) . (

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

(D) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA-mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a) or Rab6a/a' (siRab6a/a') after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (E) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock-infected or infected with wild type (2308), ΔbspF, complemented ∆bspF (ΔbspF::bspF), ∆bspB or complemented ∆bspB (ΔbspB::bspB) bacteria for 24 h, incubated for 30 min with AlexaFluor™488-CTxB on ice for binding followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to mock-infected cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (


( G - I ) Brucella replication in BMMs treated with non - targeting siRNAs ( siNT ) , or siRNAs against Rab6a / a ' ( siRab6a / a ' ) ( G ) , Rab8a ( siRab8a ) ( H ) or Arf6 ( siArf6 ) ( I ) infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) , measured as number of per at 24 h pi . Data are means ± SD of n = 3 independent experiments in which at least 100 were analyzed per experiment . Grey dots represent analyzed ; black dots indicate means of experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons ) between control conditions .

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

(G-I) Brucella replication in BMMs treated with non-targeting siRNAs (siNT), or siRNAs against Rab6a/a' (siRab6a/a') (G), Rab8a (siRab8a) (H) or Arf6 (siArf6) (I) and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


( B ) Representative co - immunoprecipitations of HA - BspF myc - ACAP1 in HeLa . HeLa were transfected to either co - produce or individually produce HA - BspF myc - ACAP1 either HA - BspF or myc - ACAP1 were immunoprecipitated using either anti - HA - conjugated ( upper panel ) or anti - myc - conjugated ( lower panel ) magnetic beads following cross - linking ( + DSP ) or not ( - DSP ) with dithiobis [ succinimidylpropionate ] . Input lysates ( 10 % of post - supernatant ) co - immunoprecipitates were separated by SDS - PAGE probed for HA - BspF myc - ACAP1 by Western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative co-immunoprecipitations of HA-BspF and myc-ACAP1 in HeLa cells. HeLa cells were transfected to either co-produce or individually produce HA-BspF and myc-ACAP1 and either HA-BspF or myc-ACAP1 were immunoprecipitated using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear supernatant) and co-immunoprecipitates were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting.


( C ) Representative confocal of HeLa transfected to produce mCherry - BspF GFP - ACAP1 treated with Cytochalasin D ( 200 nM ) for 30 min of colocalization between mCherry - BspF GFP - ACAP1 . Arrows indicate areas of BspF ACAP1 colocalization . Scale bars : 10 µm 2 µm ( insets ) . Data are means ± SD from n = 3 independent experiments in which 10 were analyzed per experiment . Pearson ' s correlation coefficients were calculated from whole using NIH Fiji image software Coloc _ 2 plug - in .

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

(C) Representative confocal micrographs of HeLa cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.


( B ) Representative Western blot of co - immunoprecipitations of myc - ACAP1 Arf6 - HA in presence or absence of HA - BspF . HeLa were transfected to produce Arf6 - HA combinations of myc - ACAP1 HA - BspF , or not , myc - ACAP1 was immunoprecipitated using anti - myc - conjugated magnetic beads . Input lysates ( 6 % of post - supernatants ) co - immunoprecipitates were separated by SDS - PAGE probed for Arf6 - HA , HA - BspF myc - ACAP1 by Western blotting . of Arf6 / ACAP1 was performed by densitometric . Data are means ± SD of 3 independent experiments . asterisk indicates a statistically significant difference ( P = 0 . 0017 , unpaired Student ' s t ) between BspF - producing control conditions .

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

(B) Representative Western blot analysis of co-immunoprecipitations of myc-ACAP1 and Arf6-HA in the presence or absence of HA-BspF. HeLa cells were transfected to produce Arf6-HA and combinations of myc-ACAP1 and HA-BspF, or not, and myc-ACAP1 was immunoprecipitated using anti-myc-conjugated magnetic beads. Input lysates (6% of post-nuclear supernatants) and co-immunoprecipitates were separated by SDS-PAGE and probed for Arf6-HA, HA-BspF and myc-ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference (P = 0.0017, unpaired Student's t test) between BspF-producing and control conditions.


( E ) replication in BMMs transduced to either produce GFP , GFP - ACAP1 or GFP - ACAP1R448Q infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) for 24 h . Data are means ± SD of n = 3 independent experiments , in which at least 100 were analyzed per experiment . Grey dots represent analyzed ( n > 300 ) ; black dots indicate means of experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons ) between control conditions .

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

(E) Bacterial replication in BMMs transduced to either produce GFP, GFP-ACAP1 or GFP-ACAP1R448Q and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


( D - F ) Recruitment of Stx6 - positive vesicles to rBCVs in BMMs treated with non - targeting siRNAs ( siNT ) , siRNAs against Arf6 ( siArf6 ) ( D ) , Rab8a ( siRab8a ) ( E ) or Rab6a / a ' ( siRab6a / a ' ) ( F ) infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) . Data are means ± SD of n = 3 independent experiments , in which 200 BCVs were analyzed per experiment . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons ) between control conditions ; ns , not significant .

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

(D-F) Recruitment of Stx6-positive vesicles to rBCVs in BMMs treated with non-targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E) or Rab6a/a' (siRab6a/a') (F) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions; ns, not significant.


( H ) Brucella replication in BMMs treated with either non - targeting siRNAs ( siNT ) , or siRNAs against Stx6 ( siStx6 ) infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) . Data are means ± SD of n = 3 independent experiments , in which at least 100 were analyzed per experiment . Grey dots represent analyzed ( n > 300 ) ; black dots indicate means of experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons ) between control conditions .

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

(H) Brucella replication in BMMs treated with either non-targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


A . Immunostaining of P5 cerebellum with indicated antibodies . ( A ) low magnification . ( A ' - A ' ' ' ' ) high magnification . solid indicates pial edge of EGL . dashed demarcates bottom of KI67 - stained region . outer EGL corresponds to between solid dashed lines , which contain GCPs .

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

A. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs.


Atoh1 KD . ( G , H ) Atoh1 shRNA was introduced to P5 mice EGL together with H2B - EGFP plasmid immunostaining for NEUROD1 ( Cyan ) KI67 ( magenta ) was performed 24 hrs later . Nuclei of electroporated were labeled with GFP ( green ) . of Control ( G ) Atoh1 KD ( H ) . Arrows indicate of interest where GFP signals are co - localized with NEUROD1 KI67 .

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

Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67.


( I ) Proportions of NEUROD1 + , KI67 + ( ND + GCPs ) among electroporated GCPs ( GFP + , KI67 + ) were increased in Atoh1 KD mice . ( J ) Proportions of KI67 + ( total GCPs ) were not significantly changed . Student T - was performed between Control ( N = 4 ) Atoh1 shRNA ( sh # 1 : N = 3 , sh # 2 : N = 3 ) . Bars error bars are representing as Mean ± . * p < 0 . 05 , N . S . : Not Significant . Number of : 98 , 71 , 81 60 from N = 4 mice ( sclamble ) , 127 , 93 51 from N = 3 mice ( sh # 1 ) , 75 , 111 , 38 from N = 3 mice ( sh # 2 ) .

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

(I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).


B . Immunostaining of P5 cerebellum with indicated antibodies . Boxplots of of CCND1 immuno - signal ( B ' ' ' ' , normalized to mean of AT + GCPs ) . We measured intensities of CCND1 in ATOH1 + / NEUROD1 - NEUROD1 + / ATOH1 - . ATOH1 + / NEUROD1 + were not included in this . These boxplots are showing median ( central band ) , 25 % / 75 % quartiles ( boxes ) ranges ( lower whiskers represent regions of lower box - 1 . 5 * IQR ( inter - quartile range ) , upper whisker represent regions of upper box - 1 . 5 * IQR ) . Wilcoxon were performed between AT + GCPs ND + GCPs . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.


C - F . Ccnd1 KD . ( C , D ) Ccnd1 shRNAs together with H2B - EGFP plasmid were introduced to P5 mice EGL immunostaining for ATOH1 ( magenta ) KI67 ( Cyan ) was performed following day . Nuclei of electroporated were labeled with GFP ( yellow ) . of Control ( C ) Ccnd1 KD ( D ) . Arrows indicate of interest where GFP signals are co - localized with ATOH1 . ( E ) Proportions of ATOH1 + , KI67 + ( AT + GCPs ) among electroporated were decreased in Ccnd1 KD mice . ( F ) Proportions of KI67 + ( total GCPs ) were not significantly changed . Student T - was performed between Control ( N = 3 ) Ccnd1 KD ( sh # 1 : N = 3 , sh # 2 : N = 3 ) mice . Bars error bars are representing as Mean ± . * * * p < 0 . 001 , N . S . : Not Significant . Number of : 52 , 64 107 from N = 3 mice ( scramble ) , 72 , 79 75 from N = 3 mice ( sh # 1 ) , 84 , 70 40 from N = 3 mice ( sh # 2 ) .

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

C-F. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2).