Figure 5B Relative expression of Pltp in liver of mice that received adenovirus GFP ( control ) or PLTP . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * P < 0 . 01 relative to GFP by ANOVA one - way followed by Tukey ' s test . N . S . , not significant . C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * * P < 0 . 001 relative to GFP by ANOVA one - way followed by Tukey ' s test . N . S . , not significant . D Plasma PLTP activity in mice that received adenovirus GFP or PLTP . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * * P < 0 . 001 PLTP relative to GFP and PLTPM159E ; * P < 0 . 05 PLTPM159E relative to GFP by ANOVA one - way followed by Tukey ' s test . E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD . n = 8 for GFP , n = 10 for PLTP and n = 10 for PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 relative to PLTPM159E by two - way ANOVA repeated measures followed by Bonferroni ' s test . F Relative Ucp1 expression in BAT of mice that received AAV GFP ( control ) or PLTP at 10 weeks of HFD . n = 4 for GFP , n = 5 for PLTP and n = 4 for PLTPM159E . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 by ANOVA one - way followed by Tukey ' s test . G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD . n = 5 for GFP and n = 6 for both PLTP and PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 PLTP relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test . AUC of glucose , shown in the right graph , was analyzed by one - way ANOVA followed by Tukey ' s test . N . S . , not significant . H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD . n = 7 for all groups . * P = 0 . 05 by ANOVA one - way followed by Fisher ' s LSD test . N . S . , not significant . I Plasma bile acids in ( H ) . n = 7 for all groups . * * * P < 0 . 001 PLTP relative to GFP and PLTPM159E by ANOVA one - way followed by Tukey ' s test . J Plasma phospholipid in ( H ) . n = 7 for all groups . * * * P < 0 . 001 PLTP and PLTPM159E relative to GFP ; * P < 0 . 05 PLTP relative to PLTPM159E by ANOVA one - way followed by Fisher ' s LSD test .

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

Figure 5B Relative expression of Pltp in liver of mice that received adenovirus GFP (control) or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. **P<0.01 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.D Plasma PLTP activity in mice that received adenovirus GFP or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 PLTP relative to GFP and PLTPM159E; *P<0.05 PLTPM159E relative to GFP by ANOVA one-way followed by Tukey's test.E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD. n=8 for GFP, n=10 for PLTP and n=10 for PLTPM159E. *P<0.05, **P<0.01 relative to PLTPM159E by two-way ANOVA repeated measures followed by Bonferroni's test.F Relative Ucp1 expression in BAT of mice that received AAV GFP (control) or PLTP at 10 weeks of HFD. n=4 for GFP, n=5 for PLTP and n=4 for PLTPM159E. Tbp was used as an internal control. *P<0.05, **P<0.01 by ANOVA one-way followed by Tukey's test.G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD. n=5 for GFP and n=6 for both PLTP and PLTPM159E. *P<0.05, **P<0.01, ***P<0.001 PLTP relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. AUC of glucose, shown in the right graph, was analyzed by one-way ANOVA followed by Tukey's test. N.S., not significant.H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD. n=7 for all groups. *P=0.05 by ANOVA one-way followed by Fisher's LSD test. N.S., not significant.I Plasma bile acids in (H). n=7 for all groups. ***P<0.001 PLTP relative to GFP and PLTPM159E by ANOVA one-way followed by Tukey's test.J Plasma phospholipid in (H). n=7 for all groups. ***P<0.001 PLTP and PLTPM159E relative to GFP; *P<0.05 PLTP relative to PLTPM159E by ANOVA one-way followed by Fisher's LSD test.


Figure 1A . Heatmap of IC50 values for cell viability . Human dermal fibroblasts ( HDFB ) controls and 4 MCC cell lines ( PeTa , MKL - 1 , WaGa , MS - 1 ) were treated with the 43 indicated small molecules targeting epigenetic modifiers . IC50 values are depicted as log10 ( IC50 ) in [ mM ] . n / a , IC50 values could not be calculated . n = 4 technical replicates . B . Dose - response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK - LSD1 or ORY - 1001 . Dose - response curves of 3 other MCC cell lines are displayed in Fig EV1A . n = 4 technical replicates . Data are represented as means ± SD . D . In vitro competition assay of the 3 MCC cell lines MKL - 2 , PeTa , and WaGa transduced with either shLSD1 . 1 , shLSD1 . 2 , shRenilla ( negative control ) , or shRPS15 ( positive control ) . Individual graphs are displayed in Fig EV1E . E . Dependency plot depicting the mean dependency of the 3 MCC cell lines PeTa , MKL - 1 , MKL - 2 of the genes targeted by the compound library in Fig 1A . A score of 0 indicates that a gene is not essential ; correspondingly - 1 is comparable to the median of all pan - essential genes . Data obtained from DepMap ; dependencies for the individual cell lines are displayed in Fig EV1F . F . Violin plot depicting the LSD1 dependency score in MCC compared to cancer types from 23 tissues , ordered according to mean dependency score . Red horizontal line depicts the median . Data obtained from DepMap RNAi screen . Blood , hematopoietic and lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system .

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

Figure 1A. Heatmap of IC50 values for cell viability. Human dermal fibroblasts (HDFB) controls and 4 MCC cell lines (PeTa, MKL-1, WaGa, MS-1) were treated with the 43 indicated small molecules targeting epigenetic modifiers. IC50 values are depicted as log10(IC50) in [mM]. n/a, IC50 values could not be calculated. n = 4 technical replicates.B. Dose-response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK-LSD1 or ORY-1001. Dose-response curves of 3 other MCC cell lines are displayed in Fig EV1A. n = 4 technical replicates. Data are represented as means ± SD.D. In vitro competition assay of the 3 MCC cell lines MKL-2, PeTa, and WaGa transduced with either shLSD1.1, shLSD1.2, shRenilla (negative control), or shRPS15 (positive control). Individual graphs are displayed in Fig EV1E.E. Dependency plot depicting the mean dependency of the 3 MCC cell lines PeTa, MKL-1, MKL-2 of the genes targeted by the compound library in Fig 1A. A score of 0 indicates that a gene is not essential; correspondingly -1 is comparable to the median of all pan-essential genes. Data obtained from DepMap; dependencies for the individual cell lines are displayed in Fig EV1F.F. Violin plot depicting the LSD1 dependency score in MCC compared to cancer types from 23 tissues, ordered according to mean dependency score. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.


Figure 2 B . Individual tumor growth in GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 22 C . Representative picture of mice treated with vehicle or GSK - LSD1 at day 41 after PeTa cell injection . Tumor location is indicated with a circle D . Kaplan - Meier curve of GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Mice were sacrificed when tumors reached a volume ≥ 1 . 5cm3 or the greatest dimension was ≥ 1 . 5cm . Red arrow : start of therapy , day 22 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox test ) F . Individual tumor growth in GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 1 G . Relative mouse weight ( % ) during treatment of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice H . Kaplan - Meier curve of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice . Mice were sacrificed when tumors reached a volume ≥ 1 . 5cm3 or greatest dimension ≥ 1 . 5cm . Red arrow : start of therapy , day 1 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox test )

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

Figure 2 B. Individual tumor growth in GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 22  C. Representative picture of mice treated with vehicle or GSK-LSD1 at day 41 after PeTa cell injection. Tumor location is indicated with a circle  D. Kaplan-Meier curve of GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or the greatest dimension was ≥ 1.5cm. Red arrow: start of therapy, day 22. ****p < 0.0001 (log-rank Mantel-Cox test)  F. Individual tumor growth in GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 1  G. Relative mouse weight (%) during treatment of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice  H. Kaplan-Meier curve of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or greatest dimension ≥ 1.5cm. Red arrow: start of therapy, day 1. ****p < 0.0001 (log-rank Mantel-Cox test)


Figure 3A . Ki - 67 staining of PeTa cells treated for 3 days with GSK - LSD1 ( 1 μM ) or ORY - 1001 ( 1 μM ) . n = 3 biological replicates . Data are represented as means ± SD . * * * p < 0 . 001 ( DMSO vs GSK - LSD1 p = 0 . 0002 ; DMSO vs ORY - 1001 p = 0 . 0004 ; unpaired Student ' s t - test ) . B . Representative FACS readout of EdU / Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK - LSD1 or DMSO treatment . C . Quantification of EdU / Propidium iodide staining depicted in Fig 3B . n = 3 biological replicates . Data are represented as means ± SD . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; ns , non - significant ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0059 ; S phase : DMSO vs GSL - LSD1 p < 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 2338 ; unpaired Student ' s t - test ) . D . Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment . Etoposide serves as positive control for apoptosis , H3 serves as loading control . c . PARP1 , cleaved PARP1 . E . Caspase - 3 / 7 cleavage activity for PeTa cell line after 6 days of indicated treatment . Etoposide serves as a positive control for apoptosis . n = 3 technical replicates . Data are represented as means ± SD . RLU , relative luminescence units . F . Representative FACS readout of tetramethylrhodamine ethyl ester ( TMRE ) stained cells upon 100 nM GSK - LSD1 or DMSO treatment after 6 days . G . Quantification of tetramethylrhodamine ethyl ester ( TMRE ) stained cells upon 100 nM GSK - LSD1 or DMSO treatment at indicated time points . H . Representative FACS readout of Annexin V / DAPI staining of PeTa cells after 6 days of 100 nM GSK - LSD1 DMSO treatment . I . Quantification of Annexin V / DAPI staining depicted in Fig 3H . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( Early , Late , Total ; unpaired Student ' s t - test ) . J . Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK - LSD1 or vehicle treatment . Representative images in Appendix Fig S1B . n = 8 , * * * p = 0 . 001 ( Vehicle vs GSK - LSD1 ; unpaired Student ' s t - test ) . ROI , region of interest . K . Top . Representative images of immunofluorescent Ki - 67 staining of tumor slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . Quantification of Ki - 67 signal of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0015 , unpaired Student ' s t - test ; D10 vehicle vs D10 GSK - LSD1 p < 0 . 0001 , unpaired Student ' s t - test ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 0012 , Mann - Whitney test ) . ROI , region of interest . L . Top . Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . Quantification of TUNEL signal of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * * p < 0 . 001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0009 ; D10 vehicle vs D10 GSK - LSD1 p = 0 . 0001 ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 001 ; unpaired Student ' s t - test with Welch ' s correction ) . ROI , region of interest . M . Representative images of hematoxylin and eosin ( H & E ) staining of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day ( D1 , top ) , 10 days ( D10 , middle ) or until experiment endpoint ( bottom ) . Upper right scale bar represents 50 μm , insert scale bar represents 10 μm .

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

Figure 3A. Ki-67 staining of PeTa cells treated for 3 days with GSK-LSD1 (1 μM) or ORY-1001 (1 μM). n = 3 biological replicates. Data are represented as means ±SD. ***p < 0.001 (DMSO vs GSK-LSD1 p = 0.0002; DMSO vs ORY-1001 p = 0.0004; unpaired Student's t-test).B. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK-LSD1 or DMSO treatment. C. Quantification of EdU/Propidium iodide staining depicted in Fig 3B. n = 3 biological replicates. Data are represented as means ±SD. **p<0.01; ****p<0.0001; ns, non-significant (G1 phase: DMSO vs GSK-LSD1 p = 0.0059; S phase: DMSO vs GSL-LSD1 p<0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.2338; unpaired Student's t-test). D. Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment. Etoposide serves as positive control for apoptosis, H3 serves as loading control. c.PARP1, cleaved PARP1.E. Caspase-3/7 cleavage activity for PeTa cell line after 6 days of indicated treatment. Etoposide serves as a positive control for apoptosis. n = 3 technical replicates. Data are represented as means ±SD. RLU, relative luminescence units.F. Representative FACS readout of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment after 6 days. G. Quantification of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment at indicated time points. H. Representative FACS readout of Annexin V/DAPI staining of PeTa cells after 6 days of 100 nM GSK-LSD1 DMSO treatment. I. Quantification of Annexin V/DAPI staining depicted in Fig 3H. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (Early, Late, Total; unpaired Student's t-test). J. Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK-LSD1 or vehicle treatment. Representative images in Appendix Fig S1B. n = 8, ***p = 0.001 (Vehicle vs GSK-LSD1; unpaired Student's t-test). ROI, region of interest.K. Top. Representative images of immunofluorescent Ki-67 staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of Ki-67 signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, **p<0.01, ****p<0.0001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0015, unpaired Student's t-test; D10 vehicle vs D10 GSK-LSD1 p<0.0001, unpaired Student's t-test; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.0012, Mann-Whitney test). ROI, region of interest.L. Top. Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of TUNEL signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, ***p<0.001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0009; D10 vehicle vs D10 GSK-LSD1 p = 0.0001; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.001; unpaired Student's t-test with Welch's correction). ROI, region of interest.M. Representative images of hematoxylin and eosin (H&E) staining of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day (D1, top), 10 days (D10, middle) or until experiment endpoint (bottom). Upper right scale bar represents 50 μm, insert scale bar represents 10 μm.


Figure 4A . Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK - LSD1 or DMSO treatment . Scale bar , 100 μm . B . Metagene plots showing the average CUT & RUN signal for H3K4me1 , H3K27ac , and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start site ; TES transcription end site . C . ATAC - seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start siteD . Volcano plot showing the - log10 ( adjusted p - value ) and log2 fold - change ( log2FC ) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK - LSD1 or DMSO for 6 days . Significantly down - regulated genes ( FDR ≤ 0 . 05 ; log2FC ≤ - 1 ) involved in mitotic cell cycle ( GO : 0000278 ) are highlighted in blue . E . Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D . Top - 5 enriched motifs are depicted . TF , transcription factor . D . Volcano plot showing the - log10 ( adjusted p - value ) and log2 fold - change ( log2FC ) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK - LSD1 or DMSO for 6 days . Significantly down - regulated genes ( FDR ≤ 0 . 05 ; log2FC ≤ - 1 ) involved in mitotic cell cycle ( GO : 0000278 ) are highlighted in blue . E . Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D . Top - 5 enriched motifs are depicted . TF , transcription factor .

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

Figure 4A. Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK-LSD1 or DMSO treatment. Scale bar, 100 μm.B. Metagene plots showing the average CUT&RUN signal for H3K4me1, H3K27ac, and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site; TES transcription end site.C. ATAC-seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start siteD. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor. D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor.


Figure 5B . MA - plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling . Significantly up - and downregulated genes ( FDR ≤ 0 . 05 ; abs ( log2FC ) ≥ 0 . 5 ) are marked in red and blue , respectively . Genes belonging to neuronal differentiation ( GO : 0048666 ) are labelled . FC , fold - change ; CMP , counts per million . H . Gene set enrichment analysis ( GSEA ) of the generated Merkel cell signature on the data in Fig 4B . NES , normalized enrichment score ; FDR , false discovery rate .

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Figure 5B. MA-plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling. Significantly up- and downregulated genes (FDR≤0.05; abs(log2FC)≥0.5) are marked in red and blue, respectively. Genes belonging to neuronal differentiation (GO:0048666) are labelled. FC, fold-change; CMP, counts per million.H. Gene set enrichment analysis (GSEA) of the generated Merkel cell signature on the data in Fig 4B. NES, normalized enrichment score; FDR, false discovery rate.


Figure 6A . Schematics and immunoblot of LSD1 co - immunoprecipitation ( coIP ) from PeTa cells . IB , Immunoblot ; MS , mass spectrometry . B . Volcano plot displaying the protein - protein binding partners of LSD1 . Proteins belonging to the LSD1 complex are marked in red , protein localization in blue and other protein interaction partners in green . FC , fold - change . C . Protein - protein interaction ( PPI ) mapping of the identified LSD1 binding partners . Individual complexes and p - values are displayed in Fig EV 6A . D . Volcano plot depicting the protein - protein binding partners of LSD1 depleting in PeTa cells treated with ORY - 1001 versus DMSO . Proteins belonging to the LSD1 complex are marked in red , protein localization in blue and other protein interaction partners in green . FC , fold - change . E . Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues , ordered according to mean . Red horizontal line depicts the median . Data obtained from DepMap RNAi screen . Blood , hematopoietic and lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system . G . Volcano plot depicting the differentially regulated proteins obtained from Fig 6F . Proteins members of the LSD1 - CoREST complex are labeled and highlighted in red . Proteins significantly upregulated ( - log10 ( p - adjusted ) ≥ 1 ; log2FC ≥ 0 . 75 ) and involved in nervous system development ( GO : 0007399 ) are labelled . FC , fold - change . H . Left . Immunoblot confirmation of HMG20B downregulation . H3 serves as nuclear fraction control and α - Tubulin as loading control . Right . Immunoblot quantification . HMG20B protein levels are normalized to H3 and relative to DMSO control . K . Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B ( shHMG20B ) or negative control ( shRenilla ) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA . Cell survival is depicted at day 8 after transduction and normalized to shRenilla . EV , empty vector control , WT , wild - type ; ΔALPHA , alpha - helices deletion ; ΔCC , coiled - coil domain deletion ; ΔHMG , HMG box deletion .

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Figure 6A. Schematics and immunoblot of LSD1 co-immunoprecipitation (coIP) from PeTa cells. IB, Immunoblot; MS, mass spectrometry.B. Volcano plot displaying the protein-protein binding partners of LSD1. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change. C. Protein-protein interaction (PPI) mapping of the identified LSD1 binding partners. Individual complexes and p-values are displayed in Fig EV 6A. D. Volcano plot depicting the protein-protein binding partners of LSD1 depleting in PeTa cells treated with ORY-1001 versus DMSO. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change.E. Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues, ordered according to mean. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.G. Volcano plot depicting the differentially regulated proteins obtained from Fig 6F. Proteins members of the LSD1-CoREST complex are labeled and highlighted in red. Proteins significantly upregulated (-log10(p-adjusted)≥1; log2FC≥0.75) and involved in nervous system development (GO:0007399) are labelled. FC, fold-change.H. Left. Immunoblot confirmation of HMG20B downregulation. H3 serves as nuclear fraction control and α-Tubulin as loading control. Right. Immunoblot quantification. HMG20B protein levels are normalized to H3 and relative to DMSO control.K. Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B (shHMG20B) or negative control (shRenilla) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA. Cell survival is depicted at day 8 after transduction and normalized to shRenilla. EV, empty vector control, WT, wild-type; ΔALPHA, alpha-helices deletion; ΔCC, coiled-coil domain deletion; ΔHMG, HMG box deletion.


Figure 7B . RT - qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A . Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control . n = 4 technical replicates . Bar graphs represent mean ± SD . C . Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A . α - Tubulin serves as loading control . pSMAD1 / 5 / 9 , phospho - SMAD1 / 5 / 9 . D . Immunoblot probing for LSD1 - CoREST complex members at the different time points and conditions depicted in Fig 7A . α - Tubulin serves as loading control . E . Representative FACS readout of EdU / Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash - out . F . Quantification of EdU / Propidium iodide staining depicted in Fig 7E . n = 3 biological replicates . Data are represented as means ± SD . * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0104 ; S phase : DMSO vs GSK - LSD1 p = 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 0062 ; unpaired Student ' s t - test ) . G . Quantification of FACS Annexin V / DAPI staining of PeTa cells 8 days after drug wash - out . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( unpaired Student ' s t - test ) . I . Subcutaneous tumor growth of pretreated cells ( untreated , n = 8 ; D1 , n = 9 ; D3 , n = 9 ; D6 , n = 4 ) Data are represented as means ± SEM . * p < 0 . 05 , * * * p < 0 . 001 ( untreated vs D1 p = 0 . 023 ; untreated vs D3 p = 0 . 023 ; untreated vs D6 p = 0 . 0007 ; Kruskal - Wallis test ) .

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Figure 7B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD.C. Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. pSMAD1/5/9, phospho-SMAD1/5/9. D. Immunoblot probing for LSD1-CoREST complex members at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out. F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test).G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test).I. Subcutaneous tumor growth of pretreated cells (untreated, n = 8; D1, n = 9; D3, n = 9; D6, n = 4) Data are represented as means ±SEM. *p<0.05, ***p<0.001 (untreated vs D1 p = 0.023; untreated vs D3 p = 0.023; untreated vs D6 p = 0.0007; Kruskal-Wallis test).


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

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


Figure 2 ( b ) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq and based on percent expressed ( size of the dots ) and average level of expression ( color intensity ) . ( g ) Oxygen consumption rate ( OCR ) was measured in HE and EHT cells ( n = 3 biological replicates with 2 - 3 technical replicates each ) and oxidative phosphorylation was assessed by extracellular flux analysis . Bar graphs show relative levels ± SEM of the indicated processes ( from 3 independent experiments ; unpaired t - tests ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( j ) Live cell imaging of HE and HSC - like cells stained with TMRE ( red ) at day 3 of subculture . Representative merged brightfield / TMRE and TMRE images are shown . Scale bars , 100 µm . Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments ( HE spindle and HE round , n = 3 biological replicates with 3 technical replicates each ; HSC - like , n = 2 biological replicates with 3 technical replicates each , Kruskal - Wallis test with multiple comparisons ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( k ) Dot plots showing gene expression levels of TCA cycle enzymes detected by scRNAseq and based on percent expressed ( size of the dots ) and average level of expression ( color intensity ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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Figure 2(b) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).(g) Oxygen consumption rate (OCR) was measured in HE and EHT cells (n=3 biological replicates with 2-3 technical replicates each) and oxidative phosphorylation was assessed by extracellular flux analysis. Bar graphs show relative levels ± SEM of the indicated processes (from 3 independent experiments; unpaired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(j) Live cell imaging of HE and HSC-like cells stained with TMRE (red) at day 3 of subculture. Representative merged brightfield/TMRE and TMRE images are shown. Scale bars, 100 µm. Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments (HE spindle and HE round, n=3 biological replicates with 3 technical replicates each; HSC-like, n=2 biological replicates with 3 technical replicates each, Kruskal-Wallis test with multiple comparisons). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(k) Dot plots showing gene expression levels of TCA cycle enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


Figure 3 ( 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 . 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 . 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 .

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Figure 3(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<0.001(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<0.001(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<0.001


Figure 4 ( 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 ) .

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Figure 4(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).


Figure 5 ( 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 . 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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<0.0001(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<0.0001(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<0.0001(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<0.0001(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).


Figure 2 ( 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 . 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 ) .

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Figure 2(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.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).


Figure 1 ( 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 ) .

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Figure 1(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).


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 ) .

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Figure 3(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).


Figure 4 ( 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 ) . ( G ) Luciferase assay using 3xSd - Luciferase reporter in S2 cells , transfected with single or combination of Yki isoforms . Error bars represent SD , n = 6 . * p - value < 0 . 05 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) . ( H ) Co - overexpression of Yki isoforms using GMR - Gal4 driver provides evidence of competition between Yki isoforms .

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Figure 4(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). (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.


Figure 5 ( 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 .

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Figure 5(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.


Figure 6 ( 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 .

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Figure 6(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.


Figure 1C . 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 .

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Figure 1C. 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.


Figure 2B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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.


Figure 3A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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.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.


Figure 4A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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.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.


Figure 5C . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C. 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.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.


Figure 6C , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C, 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.


Figure 7B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. 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 < 0.001


Figure 1 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.