F . Heat map for the variation of the ALDHbr cells proportion under drug treatment , in five different cell lines . Red and blue indicate the K - score in treated conditions , respectively , above and below the K - score in the untreated condition .

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F. Heat map for the variation of the ALDHbr cells proportion under drug treatment, in five different cell lines. Red and blue indicate the K-score in treated conditions, respectively, above and below the K-score in the untreated condition.


G - H . Primary ( n = 4 ) ( G ) and secondary ( n = 4 ) ( H ) tumorsphere - forming efficiency ( SFE ) of SUM159 cells under treated and untreated conditions . Grey dashed - line corresponds to the level of SFE in the untreated conditions . Statistical test used is Pairwise chi - square test . Data represent mean ± SD .

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G-H. Primary (n=4) (G) and secondary (n=4) (H) tumorsphere-forming efficiency (SFE) of SUM159 cells under treated and untreated conditions. Grey dashed-line corresponds to the level of SFE in the untreated conditions. Statistical test used is Pairwise chi-square test. Data represent mean ±SD.


A . Interaction surface built using the estimated combination index ( CI ) for the Salinomycin and JQ1 drug combination , in four different breast cancer cell lines ( top panels ) . The CI is represented in 2D color code interaction surface . The color code spans from strong synergism ( dark blue , CI < 1 ) to strong antagonism ( red , CI > 1 ) . The concentrations of each particular drug in the combinations are denoted on each axis . On the bottom panels , neighborhood Z - core matrix estimating the significant synergistic interaction . The color code spans from a statistically significant synergism ( dark blue , CI < 1 , p < 0 . 01 ) to a statistically significant antagonism ( dark red , CI > 1 , p < 0 . 01 ) .

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A. Interaction surface built using the estimated combination index (CI) for the Salinomycin and JQ1 drug combination, in four different breast cancer cell lines (top panels). The CI is represented in 2D color code interaction surface. The color code spans from strong synergism (dark blue, CI<1) to strong antagonism (red, CI>1). The concentrations of each particular drug in the combinations are denoted on each axis. On the bottom panels, neighborhood Z-core matrix estimating the significant synergistic interaction. The color code spans from a statistically significant synergism (dark blue, CI<1, p<0.01) to a statistically significant antagonism (dark red, CI>1, p<0.01).


B . Representative examples of flow chart for the ALDEFLUOR staining following different JQ1 / Salinomycin drug combinations . DEAB is an ALDH inhibitor used as negative control . Data represent mean ± SD .

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B. Representative examples of flow chart for the ALDEFLUOR staining following different JQ1/Salinomycin drug combinations. DEAB is an ALDH inhibitor used as negative control. Data represent mean ±SD.


C - D . Primary ( n = 4 ) ( C ) and secondary ( n = 4 ) ( D ) tumorsphere - forming efficiency ( SFE ) of SUM159 cells under mono - and combined - therapy compared to untreated conditions . Statistical test used is Pairwise chi - square test . .

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C-D. Primary (n=4) (C) and secondary (n=4) (D) tumorsphere-forming efficiency (SFE) of SUM159 cells under mono- and combined-therapy compared to untreated conditions. Statistical test used is Pairwise chi-square test..


E . Bar plots representing the dose - dependent changes in the proportion of apoptotic cells ( YO - PRO - 3 + ) in each cell subpopulations , in response to drug treatments ( right panel ) . Cisplatin ( CisPt ) is used as a positive control . On the right panel , representative fluorescence images obtained in different treatment conditions ( n = 6 ) . Nucleus are in blue ( Hoechst ) , bCSCs are in green ( ALDH ) , and apoptotic cells in red ( YO - PRO - 3 ) .

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E. Bar plots representing the dose-dependent changes in the proportion of apoptotic cells (YO-PRO-3+) in each cell subpopulations, in response to drug treatments (right panel). Cisplatin (CisPt) is used as a positive control. On the right panel, representative fluorescence images obtained in different treatment conditions (n=6). Nucleus are in blue (Hoechst), bCSCs are in green (ALDH), and apoptotic cells in red (YO-PRO-3).


G . Representation of the BFP + / RFP + cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone , or in combination . Blue bars : BFP + cells proportion , Red bars : RFP + cells proportion .

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G. Representation of the BFP+/RFP+ cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone, or in combination. Blue bars: BFP+ cells proportion, Red bars: RFP+ cells proportion.


H - I . Representation of the bCSC proportion in the BFP + ( H ) and RFP + ( I ) progenies in the control cells compared to the JQ1 - and Salinomycin - treated cells alone or in combination ( n = 4 ) . Statistical test used is a student ' s t - test . JQ1 Vs CTRL and Sal Vs JQ1 p = 0 . 03 ; JQ1 / Sal Vs CTRL p = 0 . 01 . Error bars represent mean ± SD .

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H-I. Representation of the bCSC proportion in the BFP+ (H) and RFP+ (I) progenies in the control cells compared to the JQ1- and Salinomycin-treated cells alone or in combination (n=4). Statistical test used is a student's t-test. JQ1 Vs CTRL and Sal Vs JQ1 p=0.03; JQ1/Sal Vs CTRL p=0.01. Error bars represent mean ± SD.


B . Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 ( n = 10 ) The grey area corresponds to the period of drug treatments .

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B. Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 (n=10) The grey area corresponds to the period of drug treatments.


C - E . Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle - treated tumors , measured by flow cytometry . Statistical test used is a student t - test . Data represent mean ± SD ( n = 10 ) .

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C-E. Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10).


Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle - treated tumors , measured by flow cytometry . Statistical test used is a student t - test . Data represent mean ± SD ( n = 10 ) .

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Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10).


Reimplantation assay ( CRCM434 ) . Two - week treated PDXs were reimplanted , in serial dilutions , into new recipient mice and tumor growth was monitored Each curve represents the growth kinetic from one individual injection .

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Reimplantation assay (CRCM434). Two-week treated PDXs were reimplanted, in serial dilutions, into new recipient mice and tumor growth was monitored Each curve represents the growth kinetic from one individual injection.


G - I . Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis ( ELDA ) . Results are expressed as the estimated number of bCSCs for 10 , 000 tumor cells . Statistical test used is Pairwise chi - square test .

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G-I. Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis (ELDA). Results are expressed as the estimated number of bCSCs for 10,000 tumor cells. Statistical test used is Pairwise chi-square test.


J - L . Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434 . Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs ( J ) . Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal / JQ1 treated mice compared to the control mice ( K , CRCM404 , p = 0 . 04 ; L , CRCM434 , p = 0 . 02 ) . Statistical test used is a Wilcoxon test . Error bars represent mean ± SD ( n = 10 ) .

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J-L. Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434. Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs (J). Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal/JQ1 treated mice compared to the control mice (K, CRCM404, p=0.04; L, CRCM434, p=0.02). Statistical test used is a Wilcoxon test. Error bars represent mean ± SD (n=10).


C . Kaplan - Meier relapse - free survival curve according to subnetwork metagenes expression levels in breast tumors . Curves were compared with a log - rank test ( p - value ) .

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C. Kaplan-Meier relapse-free survival curve according to subnetwork metagenes expression levels in breast tumors. Curves were compared with a log-rank test (p-value).


D Changes ( Δ g ) in inguinal WAT ( iWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant . E Changes ( Δ g ) in epididymal WAT ( eWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test . N . S . , not significant .

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D Changes (Δ g) in inguinal WAT (iWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant. E Changes (Δ g) in epididymal WAT (eWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01, ***P<0.001 relative to GFP by Student's t-test. N.S., not significant.


F Expression of Pltp mRNA in indicated tissues . Data are shown by relative Pltp mRNA levels normalized by 18s expression . n = 3 for all tissues ( except BAT and WAT n = 5 , Testis n = 2 ) . * P < 0 . 05 Pltp expression in BAT vs other tissues ( except testis and lung ) by Student ' s t - test .

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F Expression of Pltp mRNA in indicated tissues. Data are shown by relative Pltp mRNA levels normalized by 18s expression. n=3 for all tissues (except BAT and WAT n=5, Testis n=2). *P<0.05 Pltp expression in BAT vs other tissues (except testis and lung) by Student's t-test.


G Plasma PLTP activity from control ( Ppargflox / flox ) and BAT - less mice ( Pparg UCP1 - KO , Ucp1 - Cre x Ppargflox / flox ) . Following 6 - h fasting plasma was obtained from PpargUCP1 - KO mice and littermate control . n = 3 for both groups . * P < 0 . 05 relative to control mice by Student ' s t - test .

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G Plasma PLTP activity from control (Ppargflox/flox) and BAT-less mice (Pparg UCP1-KO, Ucp1-Cre x Ppargflox/flox). Following 6-h fasting plasma was obtained from PpargUCP1-KO mice and littermate control. n=3 for both groups. *P < 0.05 relative to control mice by Student's t-test.


H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type ( WT ) and aP2 - PRDM16 mice [ 24 ] . Peptide levels was determined by quantitative proteomics . n = 2 for both groups .

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H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type (WT) and aP2-PRDM16 mice [24]. Peptide levels was determined by quantitative proteomics. n=2 for both groups.


I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ . ChIP - seq data for PRDM16 and PPARγ were obtained from [ 31 , 32 ] , respectively .

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I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ. ChIP-seq data for PRDM16 and PPARγ were obtained from [31,32], respectively.


B Changes in body weight of mice in ( A ) during 10 weeks of HFD . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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B Changes in body weight of mice in (A) during 10 weeks of HFD. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05, **P<0.01, ***P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


C Changes in food intake of mice in ( A ) during 10 weeks of HFD ( cohort 1 ) . n = 9 for GFP ( cage 1 = 5 mice ; cage 2 = 4 mice ) and n = 8 for PLTP ( cage 1 = 4 mice ; cage 2 = 4 mice )

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C Changes in food intake of mice in (A) during 10 weeks of HFD (cohort 1). n=9 for GFP (cage 1 = 5 mice; cage 2 = 4 mice) and n=8 for PLTP (cage 1 = 4 mice; cage 2 = 4 mice)


D Body composition of mice in ( A ) at 10 weeks of HFD determined by EchoMRI . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 relative to GFP by two - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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D Body composition of mice in (A) at 10 weeks of HFD determined by EchoMRI. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05 relative to GFP by two-way ANOVA followed by Tukey's test. N.S., not significant.


E Whole - body energy expenditure of mice at 3 weeks of HFD . Area under the curve ( AUC ) of VO2 is shown in the right graph . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . * P < 0 . 05 relative to GFP by Student ' s t - test .

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E Whole-body energy expenditure of mice at 3 weeks of HFD. Area under the curve (AUC) of VO2 is shown in the right graph. n=7 for GFP and n=8 for PLTP (cohort 2). *P<0.05 relative to GFP by Student's t-test.


F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . N . S . , not significant by Student ' s t -

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F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis. n=7 for GFP and n=8 for PLTP (cohort 2). N.S., not significant by Student's t-test


G OCR in the interscapular BAT and iWAT of control and PLTP - treated mice . One week after the treatment , BAT and iWAT were isolated from mice ( 7 weeks old ) that received adenovirus for GFP or PLTP . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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G OCR in the interscapular BAT and iWAT of control and PLTP-treated mice. One week after the treatment, BAT and iWAT were isolated from mice (7 weeks old) that received adenovirus for GFP or PLTP. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


H Gene expression of indicated thermogenic genes in ( A ) from interscapular BAT for GFP ( n = 8 ) and PLTP ( n = 8 ) groups ( cohort 1 ) except n = 7 for Ucp1 of the PLTP group . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant .

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H Gene expression of indicated thermogenic genes in (A) from interscapular BAT for GFP (n=8) and PLTP (n=8) groups (cohort 1) except n=7 for Ucp1 of the PLTP group. Tbp was used as an internal control. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant.


I H & E staining of interscapular BAT from mice in ( A ) at 10 weeks of HFD ( cohort 1 ) . Representative images from n = 3 mice for both groups . Scale Bar , 50µm .

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I H&E staining of interscapular BAT from mice in (A) at 10 weeks of HFD (cohort 1). Representative images from n=3 mice for both groups. Scale Bar, 50µm.


A Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 10 weeks of HFD . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 9 for GFP and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


B Insulin tolerance test of mice in ( A ) at 10 weeks of HFD . Mice were fasted for four hours and administered insulin ( 0 . 75 U / kg body weight ) . n = 9 for GFP control and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


C Fasting insulin levels of mice in ( A ) at 10 weeks of HFD . Blood samples were harvested after eight hours of fasting . n = 5 for GFP and n = 4 for PLTP . * P < 0 . 05 relative to GFP by Student ' s t - test .

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C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.


D Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 6 weeks of HFD . Note that no difference was found in body weight between the groups by two - way ANOVA repeated measures followed by Bonferroni ' s test . . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 7 for GFP control and n = 10 for PLTP . AUC of glucose is shown in the right graph . * P < 0 . 05 by Student ' s t - test .

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D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test.


E 18F - FDG PET / CT scan images of mice that received adenovirus GFP ( control ) or PLTP at 14 weeks of HFD . Mice received CL316 , 243 at dose of 1 mg / kg of body weight 30 min before the PET / CT images . Representative images of n = 5 for both groups . Yellow arrows indicate anatomical references as bladder , heart and interscapular BAT .

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E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT.


F Quantification of 18F - FDG uptake in indicated tissues from mice in ( E ) . After the 18F - FDG PET / CT imaging , indicated tissues were harvested and the radioactivity was measured by scintillation counter . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


A Lipidomics - based quantification of indicated lipid species in mice that received tail - vein injection of adenovirus expressing PLTP or GFP ( control ) . Blood and tissues were harvested after 10 weeks of HFD . n = 4 for GFP and n = 5 for PLTP in mice on HFD . LPC = ( lyso ) phosphatidylcholine , LPE = ( lyso ) phosphatidylethalonamine , PA = phosphatidic acid , SM = sphingomyelin , TAG = triglycerides , FFA = free fatty acid . Statistical analysis is demonstrated ( P value ) relative to GFP by Student ' s t - test .

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

A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso)phosphatidylcholine, LPE = (lyso)phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test.


B Plasma total cholesterol levels in mice that received adenovirus GFP ( control ) or PLTP . Blood and tissues were harvested seven days after adenovirus infection . n = 8 in mice on RD and n = 7 in mice on HFD for 14 weeks . * * * P < 0 . 001 relative to GFP by Student ' s t - test .

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B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.


C Relative mRNA levels of indicated genes in the liver of mice on HFD . Tbp was used as an internal control . n = 4 for GFP and n = 6 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test .

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C Relative mRNA levels of indicated genes in the liver of mice on HFD. Tbp was used as an internal control. n=4 for GFP and n=6 for PLTP. *P<0.05, **P<0.01 relative to GFP by Student's t-test.


D Bile acid levels in the liver of mice in ( B ) . n = 8 in mice on RD and n = 6 in mice on HFD . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test .

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D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test.


E Bile acid levels in the plasma of mice in ( B ) . n = 8 in mice on RD and n = 7 in mice on HFD . * * * P < 0 . 001 relative to GFP by Student ' s t - test . F Bile acid levels in the feces of mice in ( B ) . Feces were collected from mice on RD and HFD after seven days of treatment . n = 7 in mice on RD and HFD . * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test .

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E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test. F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test.


H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid ( CA ) . Cells were treated with CA at indicated concentrations for 24 h . 0 . 1 % ETOH was used as vehicle control ( Ctr ) . n = 3 . Ucp1 mRNA expression was normalized by Tbp as an internal control . * * P < 0 . 01 relative to Ctr by one - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.


B 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 .

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B 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

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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.


B . Individual tumor growth in GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 22

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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

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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 )

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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

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

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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 )

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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)


A . 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 ) .

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A. 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 ) .

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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 .

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


E . Caspase - 3 / 7 cleavage activity for PeTa 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 .

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


F . Representative FACS readout of tetramethylrhodamine ethyl ester ( TMRE ) stained 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 .

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


H . Representative FACS readout of Annexin V / DAPI 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 ) .

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


J . 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 .

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


K . Top . Representative 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 .

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


L . Top . Representative 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 .

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


M . Representative 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 .

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


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

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


B . Metagene plots showing 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 .

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


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

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


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

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


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B . MA - plot of 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 .

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


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


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

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


B . Volcano plot displaying 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 .

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


D . Volcano plot depicting 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 .

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


E . Violin plot depicting 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 .

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


G . Volcano plot depicting 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 .

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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 .

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


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


B . RT - qPCR 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 .

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


C . Immunoblot probing for sustainability of LSD1 activity inhibition at 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 .

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


E . Representative FACS readout of EdU / Propidium iodide 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 ) .

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


G . 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 ) .

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


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


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


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

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


( g ) Oxygen consumption rate ( OCR ) was measured in HE 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 .

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


( j ) Live 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 .

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


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


( b - e ) FACS - sorted HE 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 .

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


( g ) Fold change in 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 .

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


( i - k ) Pregnant mice were injected with UK5099 or DCA at E9 . 5 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 .

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(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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(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 ) .

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


( d ) HE 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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


( A ) incorporation of 14C - labeled glycerol 3 - phosphate ( G3P ) into various lipids in 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 .

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(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 .

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(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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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 ) .

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


( D ) 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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(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 ) .

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(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 ) .

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


( D ) Interaction between Sd 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 ) .

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


( G ) Luciferase 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 ) .

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


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

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


( C ) Comparison of posterior / total 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 ) .

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


( E 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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(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 .

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

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.