( C ) HEK 293T were transfected with HA - S6K Thr 389 Glu mutant / or Flag - ATG1α WT or KI . immunoprecipitated S6K mutants were analysed by immunoblot using anti ‐ phosphospecific S6K Thr 229 ( top panel ) HA ( S6K ) antibodies ( middle panel ) . Flag ( ATG1α ) blot was completed from same lysates ( bottom panel ) . ATG1 , Autophagy ‐ specific gene 1 ; EGF , epidermal growth factor ; HA , haemagglutinin ; KI , ATG1α Lys 46 Ile ( kinase ‐ dead ) mutant ; RSK , ribosomal S6 kinase ; WT , wild type .

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

(C) HEK 293T cells were transfected with HA-S6K Thr 389 Glu mutant and/or Flag-ATG1α WT or KI. The immunoprecipitated S6K mutants were analysed by immunoblot using anti‐phosphospecific S6K Thr 229 (top panel) and HA (S6K) antibodies (middle panel). Flag (ATG1α) blot was completed from the same cell lysates (bottom panel). ATG1, Autophagy‐specific gene 1; EGF, epidermal growth factor; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; RSK, ribosomal S6 kinase; WT, wild type.


Mean ΔF of Rhod - FF iGluSnFR signals in response to a voltage ramp a 50 - ms - long step depolarization , respectively . synapse marked with * on overlaid image is further analyzed in following panels . ( P : pillar side , M : modiolar side ; Scale bar : 2 μm )

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Mean ΔF images of Rhod-FF and iGluSnFR signals in response to a voltage ramp and a 50-ms-long step depolarization, respectively. The synapse marked with * on the overlaid image is further analyzed in the following panels. (P: pillar side, M: modiolar side; Scale bar: 2 μm)


Voltage command ( top ) , corresponding whole - Ca2 + - influx ( middle ) functional responses ( bottom ) ( bandstop filtered at 33 . 3 Hz ) from Rhod - FF iGluSnFR , respectively . A modified Boltzmann function ( see Materials Methods , R2 = 0 . 81 ) was fitted to a Rhod - FF trace in response to a voltage ramp ( C - C ' ) . iGluSnFR - AUC was calculated per depolarization voltage used for a Boltzmann fit ( D ' , R2 = 0 . 92 ) . voltage of half - maximal activation ( V1 / 2 ) dynamic range ( 10 - 90 % ) of synaptic Ca2 + - influx glutamate release were calculated from fits depicted as circle bar in C ' D ' , respectively . obtained fits from a Ca2 + " hotspot " ( C ' ) from glutamate release ( D ' ) were plotted against each other in a voltage range from - 57 mV to - 17 mV in 1 mV increments . A power function was fitted to 25 % of maximum iGluSnFR - AUC ( R2 = 0 . 99 ) to obtain m - estimate . ( ruptured patch - clamp , 10 mM intracellular EGTA , 5 mM [ Ca2 + ] e ) ( See also Fig EV4 )

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Voltage command (top), corresponding whole-cell Ca2+-influx (middle) and the functional fluorescence responses (bottom) (bandstop filtered at 33.3 Hz) from Rhod-FF and iGluSnFR, respectively. A modified Boltzmann function (see Materials and Methods, R2 = 0.81) was fitted to a Rhod-FF fluorescence trace in response to a voltage ramp (C-C'). iGluSnFR-AUC was calculated per depolarization voltage and used for a Boltzmann fit (D', R2 = 0.92). The voltage of half-maximal activation (V1/2) and the dynamic range (10-90%) of synaptic Ca2+-influx and glutamate release were calculated from the fits and depicted as circle and bar in C' and D', respectively. The obtained fits from a Ca2+ "hotspot" (C') and from glutamate release (D') were plotted against each other in a voltage range from -57 mV to -17 mV in 1 mV increments. A power function was fitted to the 25% of the maximum iGluSnFR-AUC (R2 = 0.99) to obtain the m-estimate. (ruptured patch-clamp, 10 mM intracellular EGTA, 5 mM [Ca2+]e) (See also Fig EV4)


A voltage ramp was applied to obtain ΔF of Rhod - FF as a proxy synaptic Ca2 + influx . V1 / 2 of synaptic Ca2 + - influx was calculated from a modified Boltzman function ( see Materials Methods ) fitted to ΔF / F0 . ( mean ± SD , n = 55 synapses ; synapses are color coded ) ( see Appendix Fig S7 for fits ) .

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A voltage ramp was applied to obtain ΔF of Rhod-FF as a proxy synaptic Ca2+ influx. The V1/2 of synaptic Ca2+-influx was calculated from a modified Boltzman function (see Materials and Methods) fitted to ΔF/F0. (mean ± SD, n = 55 synapses; individual synapses are color coded) (see Appendix Fig S7 for individual fits).


Normalized iGluSnFR - AUC , in response to 50 - ms - long step depolarizations , same as A ( see Appendix Fig S8 for fits ) .

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Normalized iGluSnFR-AUC, in response to 50-ms-long step depolarizations, same as A (see Appendix Fig S8 for individual fits).


Left panel shows linear regression ( solid lines ) of V1 / 2 of synaptic Ca2 + - influx ( B ) , glutamate release ( C ) , threshold ( D ) dynamic range of release ( E ) as a function of position along pillar - modiolar axis . Shaded areas depict associated 95 % confidence intervals . Significance of correlation coefficients is reported by a two - p - value . Right panel shows box whisker plots of these properties of synapses grouped into pillar modiolar halves of IHCs . Box plots indicate first quartile ( 25th percentile ) , median third quartile ( 75th percentile ) with whiskers reaching from 10 - 90 % . For comparison of pillar modiolar synapses , either student ' s t - ( for normally distributed data ) or Mann - Whitney - U ( for non - normally distributed data ) was applied . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 .

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Left panel shows the linear regression analysis (solid lines) of V1/2 of synaptic Ca2+-influx (B), glutamate release (C), threshold (D) and dynamic range of release (E) as a function of position along the pillar-modiolar axis. Shaded areas depict the associated 95% confidence intervals. Significance of correlation coefficients is reported by a two-tailed p-value. Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. For comparison of pillar and modiolar synapses, either student's t-test (for normally distributed data) or Mann-Whitney-U test (for non-normally distributed data) was applied. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


Single exhibits different modes of Ca2 + - control of release . overlaid ΔF image of Rhod - FF ( red ) iGluSnFR ( green ) shows synaptic Ca2 + - influx glutamate release at three neighboring modiolar synapses ( synapses are color coded on their clusters ) . E ' relation between Ca2 + - influx glutamate release of given synapses in voltage range of - 57 to - 17 mV plotted with 1 mV increments . A power function was fitted until 25 % of normalized iGluSnFR AUC ( ΔF / F0 ) . Synapses showed different Ca2 + - dependencies ( m = 2 . 0 , 1 . 3 , 7 . 4 ; green , purple , orange ) .

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Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters). E' The relation between Ca2+-influx and glutamate release of given synapses in the voltage range of -57 to -17 mV plotted with 1 mV increments. A power function was fitted until the 25% of normalized iGluSnFR AUC(ΔF/F0). Synapses showed different Ca2+-dependencies (m = 2.0, 1.3, 7.4; green, purple, orange).


Single exhibits different modes of Ca2 + - control of release . overlaid ΔF image of Rhod - FF ( red ) iGluSnFR ( green ) shows synaptic Ca2 + - influx glutamate release at three neighboring modiolar synapses ( synapses are color coded on their clusters ) .

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Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters).


Mean glutamate release ( iGluSnFR - AUC ) as a function of depolarization voltage in three identified clusters ( mean ± , Cluster 1 : n = 27 synapses , Cluster 2 : n = 22 synapses , Cluster 3 : n = 6 synapses ) .

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Mean glutamate release (iGluSnFR-AUC) as a function of depolarization voltage in the three identified clusters (mean ± SEM, Cluster 1: n = 27 synapses, Cluster 2: n = 22 synapses, Cluster 3: n = 6 synapses).


B . Detection of ALDHbr by high content screening . algorithm first detect region of interest ( ROI ) in Hoechst channel ( rainbow areas ) . Then , it computes averaged ALDH signal in ROI to identify ALDHbr ( red ROIs ) calculate their proportion .

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B. Detection of ALDHbr cells by high content screening. The algorithm first detect nuclear region of interest (ROI) in the Hoechst channel (rainbow areas). Then, it computes the averaged ALDH signal in the ROI to identify ALDHbr cells (red nuclear ROIs) and calculate their proportion.


C - D . Example of candidate genes whose silencing modified ALDHbr proportion without inducing a massive ( viability > 20 % , B - score > │ 2 . 58 │ ) . Data represent mean ± SD ( n = 3 ) .

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C-D. Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death (cell viability>20%, B-score>│2.58│). Data represent mean ±SD (n=3).


E . Representative of high - content screening captures . ALDEFLUOR is represented in green . ALDHbr are labelled in red .

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E. Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. ALDHbr cells are labelled in red.


F . Volcano plot of hits . Scatter points represent genes , x - axis is log2 fold change of proportion of ALDHbr compared to control , y - axis is B - score . Genes targeted by a siRNA inducing a significant reduction ( green dots ; B - score > 2 . 58 , log2 FC < 1 ) or increase ( red dots ; B - score > 2 . 58 , log2 FC < 1 ) of ALDHbr proportion are highlighted .

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F. Volcano plot of the hits. Scatter points represent genes, the x-axis is the log2 fold change of the proportion of ALDHbr cells compared to the control, and the y-axis is the B-score. Genes targeted by a siRNA inducing a significant reduction (green dots; B-score>2.58, log2 FC<1) or increase (red dots; B-score>2.58, log2 FC<1) of the ALDHbr cell proportion are highlighted.


B . Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping ( right ) SFE ( left ) using 11 siRNAs pool ( 3 siRNAs / pool ) against selected target genes one scrambled siRNA pool used as control . Results for each siRNA pool ( from top to bottom ) are represented as opposite bars ( n = 3 ) . Correlations are measured using Spearman rank correlation ( R ) .

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B. Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping (right) and tumor SFE (left) using the 11 siRNAs pool (3 siRNAs/pool) against the selected target genes and one scrambled siRNA pool used as control. Results for each siRNA pool (from top to bottom) are represented as opposite bars (n=3). Correlations are measured using Spearman rank correlation (R).


C - D . Examples of dose - dependent ALDHbr variation in response to drug treatments . ALDHbr proportion in response to Tazemetostat in SUM159 ( n = 10 ) ( C ) JQ1 in SUM149 ( n = 10 ) ( D ) . horizontal red dashed - corresponds to a reduction of 50 % of ALDHbr proportion in treated conditions compared to control vertical red dashed - corresponds to target dose predicted by K - score . On bottom panel , corresponding flow charts for ALDEFLUOR . DEAB is an ALDH inhibitor used as negative control .

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C-D. Examples of dose-dependent ALDHbr cells variation in response to drug treatments. ALDHbr cell proportion in response to Tazemetostat in SUM159 (n=10) (C) and JQ1 in SUM149 (n=10) (D). The horizontal red dashed-line corresponds to a reduction of 50% of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed-line corresponds to the target dose predicted by the K-score. On the bottom panel, corresponding flow charts for the ALDEFLUOR staining. DEAB is an ALDH inhibitor used as negative control.


F . Heat map for variation of ALDHbr proportion under drug treatment , in five different lines . Red blue indicate K - score in treated conditions , respectively , above below K - score in 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 . ( n = 4 ) ( G ) secondary ( n = 4 ) ( H ) tumorsphere - forming efficiency ( SFE ) of SUM159 under treated untreated conditions . Grey dashed - corresponds to level of SFE in untreated conditions . Statistical used is Pairwise chi - square . 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 estimated combination index ( CI ) for Salinomycin JQ1 drug combination , in four different breast cancer lines ( top panels ) . CI is represented in 2D color code interaction surface . color code spans from strong synergism ( blue , CI < 1 ) to strong antagonism ( red , CI > 1 ) . concentrations of each particular drug in combinations are denoted on each axis . On bottom panels , neighborhood Z - matrix estimating significant synergistic interaction . color code spans from a statistically significant synergism ( blue , CI < 1 , p < 0 . 01 ) to a statistically significant antagonism ( 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 ALDEFLUOR 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 . ( n = 4 ) ( C ) secondary ( n = 4 ) ( D ) tumorsphere - forming efficiency ( SFE ) of SUM159 under mono - combined - therapy compared to untreated conditions . Statistical used is Pairwise chi - square . .

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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 dose - dependent changes in proportion of apoptotic ( YO - PRO - 3 + ) in each subpopulations , in response to drug treatments ( right panel ) . Cisplatin ( CisPt ) is used as a positive control . On right panel , representative obtained in different treatment conditions ( n = 6 ) . Nucleus are in blue ( Hoechst ) , bCSCs are in green ( ALDH ) , apoptotic 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 BFP + / RFP + proportions for increasing drug concentrations of either JQ1 or Salinomycin alone , or in combination . Blue bars : BFP + proportion , Red bars : RFP + 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 bCSC proportion in BFP + ( H ) RFP + ( I ) progenies in control compared to JQ1 - Salinomycin - treated alone or in combination ( n = 4 ) . Statistical used is a student ' s t - . JQ1 Vs CTRL 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 Salinomycin treatment on growth of CRCM434 ( n = 10 ) grey corresponds to 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 . of proportion of residual ALDHbr in treated PDX normalized to proportion of ALDHbr in vehicle - treated tumors , measured by flow cytometry . Statistical used is a student t - . 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).


of proportion of residual ALDHbr in treated PDX normalized to proportion of ALDHbr in vehicle - treated tumors , measured by flow cytometry . Statistical used is a student t - . 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 ( CRCM434 ) . Two - week treated PDXs were reimplanted , in serial dilutions , into new recipient mice growth was monitored Each curve represents growth kinetic from one 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 ( ELDA ) . Results are expressed as estimated number of bCSCs for 10 , 000 . Statistical used is Pairwise chi - square .

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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 Salinomycin treatments on metastasis formation of CRCM404 CRCM434 . Metastasis formation was monitored using bioluminescence imaging in treated mice livers lungs ( J ) . of normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal / JQ1 treated mice compared to control mice ( K , CRCM404 , p = 0 . 04 ; L , CRCM434 , p = 0 . 02 ) . Statistical used is a Wilcoxon . 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 in breast tumors . Curves were compared with a log - rank ( 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 - . 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 - . 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 normalized by 18s expression . n = 3 for tissues ( except BAT WAT n = 5 , Testis n = 2 ) . * P < 0 . 05 Pltp expression in BAT vs other tissues ( except testis lung ) by Student ' s t - .

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

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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 in culture conditioned medium from differentiated inguinal adipocytes of wild type ( WT ) aP2 - PRDM16 mice [ 24 ] . Peptide 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 Pltp gene by PRDM16 PPARγ . ChIP - seq data for PRDM16 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 of mice in ( A ) during 10 of HFD . n = 9 for GFP 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 .

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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 of HFD ( cohort 1 ) . n = 9 for GFP ( cage 1 = 5 mice ; cage 2 = 4 mice ) 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 composition of mice in ( A ) at 10 of HFD determined by EchoMRI . n = 9 for GFP n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 relative to GFP by two - way ANOVA followed by Tukey ' s . 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 - energy expenditure of mice at 3 of HFD . under curve ( AUC ) of VO2 is shown in right graph . n = 7 for GFP n = 8 for PLTP ( cohort 2 ) . * P < 0 . 05 relative to GFP by Student ' s t - .

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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 of HFD as determined by metabolic cage . n = 7 for GFP 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 interscapular BAT iWAT of control PLTP - treated mice . One week after treatment , BAT iWAT were isolated from mice ( 7 old ) that received adenovirus for GFP or PLTP . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - . 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 ) PLTP ( n = 8 ) groups ( cohort 1 ) except n = 7 for Ucp1 of PLTP group . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - . 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 of interscapular BAT from mice in ( A ) at 10 of HFD ( cohort 1 ) . Representative 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 of mice that received AAV control ( GFP ) or PLTP at 10 of HFD . After eight hours of fasting , 2 g / kg of glucose was administered . n = 9 for GFP n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s .

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

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

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

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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 of mice that received adenovirus GFP ( control ) or PLTP at 14 of HFD . Mice received CL316 , 243 at dose of 1 mg / kg of 30 min before PET / CT . Representative of n = 5 for both groups . Yellow arrows indicate anatomical references as bladder , heart 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 of 18F - FDG uptake in indicated tissues from mice in ( E ) . After 18F - FDG PET / CT imaging , indicated tissues were harvested radioactivity was measured by scintillation counter . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - . 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 - of indicated lipid species in mice that received tail - vein injection of adenovirus expressing PLTP or GFP ( control ) . Blood tissues were harvested after 10 of HFD . n = 4 for GFP 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 is demonstrated ( P value ) relative to GFP by Student ' s t - .

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

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

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

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

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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 ) . 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 . 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 n = 6 for PLTPM159E . * * P < 0 . 01 relative to GFP by ANOVA one - way followed by Tukey ' s . N . S . , not significant .

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

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 in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry . n = 4 for GFP , n = 6 for PLTP n = 6 for PLTPM159E . * * * P < 0 . 001 relative to GFP by ANOVA one - way followed by Tukey ' s . N . S . , not significant .

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

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 n = 6 for PLTPM159E . * * * P < 0 . 001 PLTP relative to GFP PLTPM159E ; * P < 0 . 05 PLTPM159E relative to GFP by ANOVA one - way followed by Tukey ' s .

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

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 of mice that received AAV control or PLTP during 10 of HFD . n = 8 for GFP , n = 10 for PLTP n = 10 for PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 relative to PLTPM159E by two - way ANOVA repeated measures followed by Bonferroni ' s .

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

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 of HFD . n = 4 for GFP , n = 5 for PLTP 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 .

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

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 of mice that received AAV GFP or PLTP at 10 of HFD . n = 5 for GFP n = 6 for both PLTP PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 PLTP relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s . AUC of glucose , shown in right graph , was analyzed by one - way ANOVA followed by Tukey ' s . N . S . , not significant .

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

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 of HFD . n = 7 for groups . * P = 0 . 05 by ANOVA one - way followed by Fisher ' s LSD . N . S . , not significant .

O B-TISSUE O B-SMALL_MOLECULE O B-ORGANISM O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 in ( H ) . n = 7 for groups . * * * P < 0 . 001 PLTP relative to GFP PLTPM159E by ANOVA one - way followed by Tukey ' s .

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

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 groups . * * * P < 0 . 001 PLTP PLTPM159E relative to GFP ; * P < 0 . 05 PLTP relative to PLTPM159E by ANOVA one - way followed by Fisher ' s LSD .

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

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 . of IC50 values for viability . Human dermal fibroblasts ( HDFB ) controls 4 MCC lines ( PeTa , MKL - 1 , WaGa , MS - 1 ) were treated with 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 .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-DISEASE O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

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 control HDFB after 6 days of treatment with GSK - LSD1 or ORY - 1001 . Dose - response curves of 3 other MCC lines are displayed in Fig EV1A . n = 4 technical replicates . Data are represented as means ± SD .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O

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 of 3 MCC lines MKL - 2 , PeTa , WaGa transduced with either shLSD1 . 1 , shLSD1 . 2 , shRenilla ( negative control ) , or shRPS15 ( positive control ) . graphs are displayed in Fig EV1E .

O O O O B-EXP_ASSAY O O B-DISEASE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 mean dependency of 3 MCC lines PeTa , MKL - 1 , MKL - 2 of genes targeted by compound library in Fig 1A . A score of 0 indicates that a gene is not essential ; correspondingly - 1 is comparable to median of pan - essential genes . Data obtained from DepMap ; dependencies for lines are displayed in Fig EV1F .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 LSD1 dependency score in MCC compared to cancer types from 23 tissues , ordered according to mean dependency score . Red horizontal depicts median . Data obtained from DepMap RNAi screen . Blood , hematopoietic lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system .

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

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

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

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 injection . location is indicated with a circle

O O O B-EXP_ASSAY O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_LINE O O B-EXP_ASSAY O O O O O

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 greatest dimension was ≥ 1 . 5cm . Red arrow : start of therapy , day 22 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox )

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

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 . 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 ( % ) during treatment of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice

O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM

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 )

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

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 of PeTa 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 - ) .

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

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 cycle of PeTa after 6 days of 100 nM GSK - LSD1 or DMSO treatment . C . of EdU / Propidium iodide 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 - ) .

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

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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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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 of glycolytic enzymes detected by scRNAseq on expressed ( of dots ) average level of expression ( color ) .

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


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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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