( 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 .

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

(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 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 .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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

(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 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 ) .

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

(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 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 .

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ± 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 .

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

(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 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 .

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

(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 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 ) .

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

(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 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 .

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

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

(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 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 ) .

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

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 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 ) .

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

(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 ) 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 .

O O O O O B-TISSUE O O B-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 ) 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 ) .

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

(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 the normalized co - IP ( NcoIP ) results for 13 proteins tested against the two Yki isoforms and the negative control ( Flag - Firefly ) .

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

(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 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 ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

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

(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 .

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

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


( 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 ) .

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

(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 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 .

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

(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 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 .

O O O O O B-TISSUE B-EXP_ASSAY O B-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 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 .

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

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

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

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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

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

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

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

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

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

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

O O O O O O O B-TISSUE O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O 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 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 .

O O O B-ORGANISM O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

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

(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 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 .

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

(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 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 ) . (

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

(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 ) 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 ) . (

O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O B-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 ) 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 .

O O O O O O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O B-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O B-CELL_TYPE 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 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 .

B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM B-TISSUE O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD 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 + 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 ) .

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

O O B-EXP_ASSAY O O B-TISSUE O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE 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 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 ) .

O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM 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).


G - K . Over - expression analysis of Ccnd1 with or without Cdk4 . ( G , H , I ) Ccnd1 and Cdk4 expression plasmids 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 ( G ) , CCND1 over expression ( H ) and CCND1 and CDK4 over expression ( I ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( J ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were not significantly changed between Control and CCND1 . Forced expression of CCND1 and CDK4 ( 4D ) significantly increased the proporations of AT + GCPs compared to Control . ( K ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Tukey - Kramer test was performed on Control ( N = 6 ) , CCND1 ( N = 4 ) , CCND1 and CDK4 ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , N . S . : Not Significant . Number of cells : 52 , 64 , 107 , 244 , 127 and 146 from N = 6 mice ( Control ) , 126 , 137 , 80 and 336 from N = 4 mice ( Ccnd1 ) , 212 , 143 and 38 from N = 3 mice ( 4D ) .

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

G-K. Over-expression analysis of Ccnd1 with or without Cdk4. (G, H, I) Ccnd1 and Cdk4 expression plasmids 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 (G), CCND1 over expression (H) and CCND1 and CDK4 over expression (I). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (J) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly increased the proporations of AT+GCPs compared to Control. (K) Proportions of KI67+ cells (total GCPs) were not significantly changed. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, N.S.: Not Significant. Number of cells: 52, 64, 107,244,127 and 146 from N=6 mice (Control), 126, 137, 80 and 336 from N=4 mice (Ccnd1), 212, 143 and 38 from N=3 mice (4D).


C . N2a cells transfected with Atoh1 - GST plasmids were treated with DMSO ( Control ) , Palbociclib ( Palb ) or Palb and 10 uM MG132 ( MG132 ) for 6hrs before harvest . Lysates of N2a were subjected to immunoblotting with anti - Atoh1 or β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between Control , Palb , MG132 . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * * p < 0 . 001 , * p < 0 . 05 , N . S . : Not Significant . Three slots of each experimental condition represent the three biological replicants of each condition .

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

C. N2a cells transfected with Atoh1-GST plasmids were treated with DMSO(Control), Palbociclib (Palb) or Palb and 10 uM MG132 (MG132) for 6hrs before harvest. Lysates of N2a were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between Control, Palb, MG132. Bars and error bars are representing as Mean ± SEM from three trials of each condition. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Three slots of each experimental condition represent the three biological replicants of each condition.


G . Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids ( 4D ) . N2a cells were treated with 10 nM , 100 nM , 1000 nM Palb for 6 hrs before harvest . Lysates from N2a cells were subjected to immunoblotting with anti - Atoh1 , p - S309 - Ab and β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between 0 , 10 , 100 , 1000nM . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant .

O O B-GENEPROD O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids (4D). N2a cells were treated with 10 nM, 100 nM, 1000 nM Palb for 6 hrs before harvest. Lysates from N2a cells were subjected to immunoblotting with anti-Atoh1, p-S309-Ab and β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between 0, 10, 100, 1000nM. Bars and error bars are representing as Mean ± SEM from three trials of each condition. **p < 0.01, *p < 0.05, N.S.: Not Significant.


B . Lysates of N2a cells transfected with either WT or a phospho - mimic form of ATOH1 ( S309D ) were immunoblotted with anti - Atoh1 or β - actin . Quantification of relative levels of ATOH1 to the β - ACTIN ( right panel ) . Mean ± s . e . m . Student T - test between WT and S309D was performed . * * * p < 0 . 001 . Three slots of each experimental condition represent the three biological replicants of each condition .

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

B. Lysates of N2a cells transfected with either WT or a phospho-mimic form of ATOH1 (S309D) were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. ***p < 0.001. Three slots of each experimental condition represent the three biological replicants of each condition.


Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1 . ( F - I ) Scramble ( Control ) , Atoh1 shRNA ( Atoh1 KD ) , shRNA and WT ( KD + WT ) or shRNA and S309A ( KD + S309A ) plasmids were introduced to P5 mice EGL with H2B - EGFP plasmid and immunostaining for NEUROD1 ( magenta ) was performed at P7 . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest which GFP signals are not co - localized with NEUROD1 ( AT + GCPs ) .

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

Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (F-I) Scramble (Control), Atoh1 shRNA (Atoh1 KD), shRNA and WT (KD + WT) or shRNA and S309A (KD + S309A) plasmids were introduced to P5 mice EGL with H2B-EGFP plasmid and immunostaining for NEUROD1(magenta) was performed at P7. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest which GFP signals are not co-localized with NEUROD1 (AT+GCPs).


B - D . Overexpression analysis of PROX1 . ( B , C ) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of Control ( B ) , PROX1 overexpression ( C ) . Arrow heads indicates the CCND1 positive cells . White lines are representing as pial surfaces . ( D ) The proportion of CCND1 - positive cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and PROX1 overexpression ( N = 3 ) . * * p < 0 . 01 . Number of cells 382 , 147 and 88 cells from N = 3 mice ( control ) , 187 , 108 and 94 cells from N = 3 mice ( Prox1 OE ) .

O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O

B-D. Overexpression analysis of PROX1. (B, C) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of Control (B), PROX1 overexpression (C). Arrow heads indicates the CCND1 positive cells. White lines are representing as pial surfaces. (D) The proportion of CCND1-positive cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and PROX1 overexpression (N=3). **p < 0.01. Number of cells 382, 147 and 88 cells from N=3 mice (control), 187, 108 and 94 cells from N=3 mice (Prox1 OE).