a , COS7 cells co - transfected with GFP - ATG14 and DsRed - ER or RFP - SEC61b ; ( an ER marker ) were immunostained for the mitochondria marker TOMM20 . Scale bars , 2 µm .

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a, COS7 cells co-transfected with GFP-ATG14 and DsRed-ER or RFP-SEC61b; (an ER marker) were immunostained for the mitochondria marker TOMM20. Scale bars, 2 µm.


b , An immunogold electron micrograph of starved HeLa cells expressing GFP - ATG14 probed with anti - GFP antibodies ( red arrows ) . Scale bar , 200 nm .

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b, An immunogold electron micrograph of starved HeLa cells expressing GFP-ATG14 probed with anti-GFP antibodies (red arrows). Scale bar, 200 nm.


c , Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP - DFCP1 . Cyto , cytosol ; micro , microsome ; mito , mitochondria ; PNS , post - nuclear supernatant .

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c, Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP-DFCP1. Cyto, cytosol; micro, microsome; mito, mitochondria; PNS, post-nuclear supernatant.


a , COS7 cells stably expressing yellow fluorescent protein ( YFP ) - ATG5 were transfected with RFP - SEC61b ; and teal fluorescent protein ( TFP ) - mito , and immunostained for TOMM20 .

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a, COS7 cells stably expressing yellow fluorescent protein (YFP)-ATG5 were transfected with RFP-SEC61b; and teal fluorescent protein (TFP)-mito, and immunostained for TOMM20.


b , Time - lapse images of COS7 cells transfected with YFP - ATG5 , RFP - SEC61b ; and TFP - mito under starved conditions .

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b, Time-lapse images of COS7 cells transfected with YFP-ATG5, RFP-SEC61b; and TFP-mito under starved conditions.


c , d , Distribution of distances of YFP - ATG5 dots fromthe centre to the surface of the ER ( c ) and to the surface of mitochondria ( d ) . e , f , Distances of YFP - ATG5 dots over their lifetime for data from c ( e ) and d ( f ) , respectively . All scale bars , 2 µm .

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c, d, Distribution of distances of YFP-ATG5 dots fromthe centre to the surface of the ER (c) and to the surface of mitochondria (d). e, f, Distances of YFP-ATG5 dots over their lifetime for data from c (e) and d (f), respectively. All scale bars, 2 µm.


a , COS7 cells expressing GFP - SEC61b ; and STX17 were immunostained for TOMM20 .

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a, COS7 cells expressing GFP-SEC61b; and STX17 were immunostained for TOMM20.


b , HeLa cells expressing GFP - ATG14 were immunostained for STX17 .

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b, HeLa cells expressing GFP-ATG14 were immunostained for STX17.


c , Immunoblot of subcellular fractions of HEK293 cells .

c, Immunoblot of subcellular fractions of HEK293 cells.


d , STX17 knockdown COS7 cells expressing GFP - ATG14 and RFP - SEC61b ; were immunostained for TOMM20 . Error bars denote s . d .

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d, STX17 knockdown COS7 cells expressing GFP-ATG14 and RFP-SEC61b; were immunostained for TOMM20. Error bars denote s.d.


e , Immuoblot of subcellular fractions of STX17 knockdown ( using two STX17 siRNAs , STX17 - 1 and STX17 - 2 ) HEK293 cells expressing GFP - DFCP1 .

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e, Immuoblot of subcellular fractions of STX17 knockdown (using two STX17 siRNAs, STX17-1 and STX17-2) HEK293 cells expressing GFP-DFCP1.


f , Co - immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag - ATG14 and Myc - STX17 .

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f, Co-immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag-ATG14 and Myc-STX17.


g , Pull - down of ATG14 complex from HeLa cells expressing Myc - STX17 or Myc . Scale bars , 5 µm ( a , b ) and 2 µm ( d ) .

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g, Pull-down of ATG14 complex from HeLa cells expressing Myc-STX17 or Myc. Scale bars, 5 µm (a, b) and 2 µm (d).


a , Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP - LC3 ( white arrows ) infected with GAS ( red arrows ) . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

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a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP-LC3 (white arrows) infected with GAS (red arrows). DAPI, 4′,6-diamidino-2-phenylindole.


b , Electron micrographs of STX17 knockdown cells . White arrows denote autolysosomes , black arrows denote autophagosome , white arrowheads mark isolation membranes .

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b, Electron micrographs of STX17 knockdown cells. White arrows denote autolysosomes, black arrows denote autophagosome, white arrowheads mark isolation membranes.


c , Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP - LC3 .

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c, Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP-LC3.


d , GFP - ATG5 dots ( arrows ) in STX17 knockdown HeLa cells . e , Quantification of staining in d .

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d, GFP-ATG5 dots (arrows) in STX17 knockdown HeLa cells. e, Quantification of staining in d.


f , Immunoblot of p62 in STX17 knockdown cells . Tubulin was used as a loading control .

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f, Immunoblot of p62 in STX17 knockdown cells. Tubulin was used as a loading control.


g , Degradation of long - lived proteins in STX17 knockdown cells . St , starved ; wor , wortmannin . All error bars denote s . d . Scale bars , 1 µm ( a , b ) , 200 nm ( c ) and 5 µm ( d ) .

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g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 µm (a, b), 200 nm (c) and 5 µm (d).


( A ) Frequency of CHD1 gene mutation ( green ) deep deletion ( blue ) or amplification ( red ) in prostate cancer patients .

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(A) Frequency of CHD1 gene mutation (green) deep deletion (blue) or amplification (red) in prostate cancer patients.


( B ) CHD1 is recruited to an I - SceI - induced DSB site and is co - localized with γH2AX . Immunofluorescence studies using U2OS19 ptight13 GFP - LacR cells containing a stably integrated I - SceI cleavage site flanked by 256 copies of lac operator ( lacO ) on one side and 96 copies of the tetracycline response element on the other side ( tetO ) . The localization of the GFP - lac repressor protein ( GFP - LacR ) at the lac - operator DNA sequences in the nucleus before ( − I - SceI ) and 16 h after I - SceI - induced ( + I - SceI ) DSB . After 16 h of doxy treatment , CHD1 and γH2AX co - localized at I - SceI cleavage site , along with DNA - bound GFP - LacR but not in uninduced cells ( − I - SceI ) . Scale bar 10 µm .

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(B) CHD1 is recruited to an I-SceI-induced DSB site and is co-localized with γH2AX. Immunofluorescence studies using U2OS19 ptight13 GFP-LacR cells containing a stably integrated I-SceI cleavage site flanked by 256 copies of lac operator (lacO) on one side and 96 copies of the tetracycline response element on the other side (tetO). The localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (− I-SceI) and 16 h after I-SceI-induced (+ I-SceI) DSB. After 16 h of doxy treatment, CHD1 and γH2AX co-localized at I-SceI cleavage site, along with DNA-bound GFP-LacR but not in uninduced cells (−I-SceI). Scale bar 10 µm.


( C ) Quantification of co - localization of CHD1 with the lac array , mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD .

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(C) Quantification of co-localization of CHD1 with the lac array, mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD.


( D ) PC3 cells were treated with NCS ( 100 ng / ml ) for 2 h and with EdU for 45 min . Cells were stained with EdU first and then proximity ligation assay ( PLA ) was performed using γH2AX and CHD1 antibodies . Scale bar 10 µm .

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(D) PC3 cells were treated with NCS (100 ng/ml) for 2 h and with EdU for 45 min. Cells were stained with EdU first and then proximity ligation assay (PLA) was performed using γH2AX and CHD1 antibodies. Scale bar 10 µm.


( E ) Quantification of PLA signal from ( D ) in EdU - positive and - negative cells using ImageJ . For quantification more than 100 cells were analyzed for each condition and represented as mean value ( n = 3 , ± SD ) .

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(E) Quantification of PLA signal from (D) in EdU-positive and -negative cells using ImageJ. For quantification more than 100 cells were analyzed for each condition and represented as mean value (n=3, ± SD).


( F ) CHD1 is recruited to the chromatin upon DNA double strand break induction . PC3 cells with stable control ( shCont ) or CHD1 shRNA ( shCHD1 ) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies . H2B was used as a loading control . See also Figure EV1A - EV1F .

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(F) CHD1 is recruited to the chromatin upon DNA double strand break induction. PC3 cells with stable control (shCont) or CHD1 shRNA (shCHD1) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies. H2B was used as a loading control. See also Figure EV1A-EV1F.


( A , B ) BHP1 or PC3 cells with stable expression of either control ( shCont ) or CHD1 shRNA ( shCHD1 ) were treated with γ - radiation ( 3 Gy ) and after 1 h and 24 h cells were immunostained for γH2AX ( A ) and the number of γH2AX foci per cell were determined for each time point , more than 50 cells were counted in each condition ( B ) . Scale bar 5 µm . The mean values of three independent experiments ( ± SD ) are shown . p - values ( 0 . 0008 and 0 . 006 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) were calculated using ANOVA .

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(A, B) BHP1 or PC3 cells with stable expression of either control (shCont) or CHD1 shRNA (shCHD1) were treated with γ-radiation (3 Gy) and after 1 h and 24 h cells were immunostained for γH2AX (A) and the number of γH2AX foci per cell were determined for each time point, more than 50 cells were counted in each condition (B). Scale bar 5 µm. The mean values of three independent experiments (± SD) are shown. p-values (0.0008 and 0.006, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA.


( C ) CHD1 depleted cells show prolonged γH2AX accumulation . PC3 cells with shCont or shCHD1 were treated with γ - radiation as in ( A ) and total protein was analyzed by Western blot .

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(C) CHD1 depleted cells show prolonged γH2AX accumulation. PC3 cells with shCont or shCHD1 were treated with γ-radiation as in (A) and total protein was analyzed by Western blot.


( D ) CHD1 - depleted cells show increased sensitivity to γ - radiation . For colony formation assay , both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ - radiation and surviving fractions were measured by counting colonies after 3 weeks , mean values are represented in the plot ( n = 3 , ± SD ) . Data were normalized to the plating efficiency . p - values ( 0 . 0009 and 0 . 002 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) were calculated using ANOVA . See also Figure EV2A - EV2B .

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(D) CHD1-depleted cells show increased sensitivity to γ-radiation. For colony formation assay, both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ-radiation and surviving fractions were measured by counting colonies after 3 weeks, mean values are represented in the plot (n=3, ± SD). Data were normalized to the plating efficiency. p-values (0.0009 and 0.002, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. See also Figure EV2A-EV2B.


( A , B ) HeLa cells harboring single copies of HR ( pGC ) or NHEJ ( pEJ ) repair substrates were transfected with either negative control ( siCont ) or siRNAs targeting CHD1 , RAD51 or XRCC4 . After 24 h of transfection , DSB was induced by transfecting cells with I - Sce - I - expressing vector ( pCMV - I - SceI - 3xNLS ) . After 48 h of transfection , GFP - positive cells were measured by flow cytometry . HR ( A ) or NHEJ ( B ) efficiency was calculated based on the fraction of GFP - positive cells and represented as mean values from three independent experiments as ± SD , 50 , 000 cells were counted for each condition .

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(A, B) HeLa cells harboring single copies of HR (pGC) or NHEJ (pEJ) repair substrates were transfected with either negative control (siCont) or siRNAs targeting CHD1, RAD51 or XRCC4. After 24 h of transfection, DSB was induced by transfecting cells with I-Sce-I-expressing vector (pCMV-I-SceI-3xNLS). After 48 h of transfection, GFP-positive cells were measured by flow cytometry. HR (A) or NHEJ (B) efficiency was calculated based on the fraction of GFP-positive cells and represented as mean values from three independent experiments as ± SD, 50,000 cells were counted for each condition.


( C ) CHD1 - depleted cells show decreased RAD51 foci after DNA damage induction . The shCont or shCHD1 PC3 cells were irradiated and co - immunostained with γH2AX and RAD51 antibodies after the indicated time points . Scale bar 5 µm . ( D ) The number of RAD51 foci co - localized with γH2AX per cell ( from C ) were counted ( n = 50 ) and represented in a graph . The data is represented in mean value of three independent experiments as ± SD , more than 50 cells were counted for each condition . See also Figure EV3A - EV3F .

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(C) CHD1-depleted cells show decreased RAD51 foci after DNA damage induction. The shCont or shCHD1 PC3 cells were irradiated and co-immunostained with γH2AX and RAD51 antibodies after the indicated time points. Scale bar 5 µm. (D) The number of RAD51 foci co-localized with γH2AX per cell (from C) were counted (n=50) and represented in a graph. The data is represented in mean value of three independent experiments as ± SD, more than 50 cells were counted for each condition. See also Figure EV3A-EV3F.


( A ) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment . Scale bar 5 µm .

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(A) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment. Scale bar 5 µm.


( B ) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP , RPA1 and RAD51 . H2B is shown as a loading control .

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(B) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP, RPA1 and RAD51. H2B is shown as a loading control.


CHD1 depletion leads to decreased ( C ) CtIP and ( D ) RPA1 recruitment to I - SceI - induced DSB in U2OS19 ptight13 GFP - LacR cells . CHD1 was depleted in U2OS19 ptight13 GFP - LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co - immunostained for γH2AX and CtIP or RPA1 . The number of cells with CtIP or RPA1 foci from ( C ) and ( D ) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD ( n = 3 ) , more than 50 cells were counted . Scale bar 5 µm .

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CHD1 depletion leads to decreased (C) CtIP and (D) RPA1 recruitment to I-SceI-induced DSB in U2OS19 ptight13 GFP-LacR cells. CHD1 was depleted in U2OS19 ptight13 GFP-LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co-immunostained for γH2AX and CtIP or RPA1. The number of cells with CtIP or RPA1 foci from (C) and (D) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD (n=3), more than 50 cells were counted. Scale bar 5 µm.


( E ) PC3 cells were transfected either with mock or siCHD1 followed by empty vector , wt mChd1 ( Wt ) or ATPase - mutant ( Mt ) mChd1 . After 48 h of transfection cells were treated with of 4 - OH tamoxifen ( 4 - OHT ) for 24 h and processed for PLA with γH2AX and CtIP antibodies . Cells demonstrating positive focal interactions indicative of DNA repair hubs ( punctate staining ) are present only in control and ( Wt ) CHD1 - rescued cells but not following knockdown or reconstitution of an ATPase - mutated CHD1 ( Mt ) . Scale bar 20 µm .

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(E) PC3 cells were transfected either with mock or siCHD1 followed by empty vector, wt mChd1 (Wt) or ATPase-mutant (Mt) mChd1. After 48 h of transfection cells were treated with of 4-OH tamoxifen (4-OHT) for 24 h and processed for PLA with γH2AX and CtIP antibodies. Cells demonstrating positive focal interactions indicative of DNA repair hubs (punctate staining) are present only in control and (Wt) CHD1-rescued cells but not following knockdown or reconstitution of an ATPase-mutated CHD1 (Mt). Scale bar 20 µm.


( F ) PC3 cells which stably express HA - mChd1 - ERT2 , were transfected with either mock or siCHD1 . After 24 h of transfection cells were treated with 4 - OHT for 24 h to induce HA - mChd1 - ERT2 nuclear translocation . Western blot analysis of chromatin fractions for CHD1 , ERT2 ( mChd1 ) , CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ . The relative quantification for the indicated proteins is shown in the graph . See also Figure EV4A - EV4P .

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(F) PC3 cells which stably express HA-mChd1-ERT2, were transfected with either mock or siCHD1. After 24 h of transfection cells were treated with 4-OHT for 24 h to induce HA-mChd1-ERT2 nuclear translocation. Western blot analysis of chromatin fractions for CHD1, ERT2 (mChd1), CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ. The relative quantification for the indicated proteins is shown in the graph. See also Figure EV4A-EV4P.


( A ) CHD1 recruitment is dependent on MRE11 activity . PC3 cells were transfected with mock or siMRE11 ( SmartPool ) and 48 h after transfection cells were treated with NCS and chromatin fractions were isolated and analyzed by Western blot .

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(A) CHD1 recruitment is dependent on MRE11 activity. PC3 cells were transfected with mock or siMRE11 (SmartPool) and 48 h after transfection cells were treated with NCS and chromatin fractions were isolated and analyzed by Western blot.


( B ) CHD1 is upstream of CtIP . PLA with γH2AX and CHD1 antibodies in mock or CtIP depleted cells after 2 h of NCS . Scale bar is 50 µm . Verification of knockdown efficiency is shown by Western blot on the right . HSC70 is shown as a loading control . Scale bar 20 µm . ( C ) Quantification of PLA signal from ( B ) represented as mean values from three independent experiments as ± SD , more than 100 cells were counted per condition .

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(B) CHD1 is upstream of CtIP. PLA with γH2AX and CHD1 antibodies in mock or CtIP depleted cells after 2 h of NCS. Scale bar is 50 µm. Verification of knockdown efficiency is shown by Western blot on the right. HSC70 is shown as a loading control. Scale bar 20 µm. (C) Quantification of PLA signal from (B) represented as mean values from three independent experiments as ± SD, more than 100 cells were counted per condition.


( D ) qPCR analyses for chromatin accessibility at two HR - repaired sites ( DSB - I and DSB - II ) was analyzed by FAIRE in AsiSI - ER - U20S cells transfected with either mock or CHD1 siRNA . After 48 h of transfection cells were treated with 4 - OHT for the indicated time points and processed for FAIRE . The data are represented as mean ± SD ( n = 3 ) p - values were calculated using ANOVA ( 0 . 02 and 0 . 03 , * p ≤ 0 . 1 ) .

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(D) qPCR analyses for chromatin accessibility at two HR-repaired sites (DSB-I and DSB-II) was analyzed by FAIRE in AsiSI-ER-U20S cells transfected with either mock or CHD1 siRNA. After 48 h of transfection cells were treated with 4-OHT for the indicated time points and processed for FAIRE. The data are represented as mean ± SD (n=3) p-values were calculated using ANOVA (0.02 and 0.03, *p ≤ 0.1).


( E ) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 ( SmartPool ) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti - BrdU and γH2AX antibodies . Scale bar 5 µm .

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(E) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 (SmartPool) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti-BrdU and γH2AX antibodies. Scale bar 5 µm.


( F ) Quantification of Brdu positive cells from ( E ) . The data are represented as mean ± SD from three independent experiments , more than 50 cells were counted for each condition . p - values ( 0 . 03 , * p ≤ 0 . 1 ) were calculated using ANOVA . See also Figure EV5A - EV4J .

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(F) Quantification of Brdu positive cells from (E). The data are represented as mean ± SD from three independent experiments, more than 50 cells were counted for each condition. p-values (0.03, *p ≤ 0.1) were calculated using ANOVA. See also Figure EV5A-EV4J.


( A ) CHD1 - depleted cells show hypersensitivity to Mitomycin ( MMC ) treatment . For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented ( n = 3 , ± SD , p - value = 0 . 0002 , ANOVA , * * * p ≤ 0 . 001 ) .

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(A) CHD1-depleted cells show hypersensitivity to Mitomycin (MMC) treatment. For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented (n=3, ± SD, p-value=0.0002, ANOVA, ***p ≤ 0.001).


( B ) CHD1 - depleted cells show increased hypersensitivity to Irinotecan . For cell proliferation analysis , shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency ( mean ) was plotted in the graph ( n = 3 , ± SD , p - value = 0 . 0004 , ANOVA , * * * p ≤ 0 . 001 ) .

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(B) CHD1-depleted cells show increased hypersensitivity to Irinotecan. For cell proliferation analysis, shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency (mean) was plotted in the graph (n=3, ± SD, p-value=0.0004, ANOVA, ***p ≤ 0.001).


( C ) Loss of CHD1 leads to increased sensitivity to PARP inhibition . Control or CHD1 - depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks . Data represented as mean values ( n = 3 , ± SD , p - value = 0 . 0003 , ANOVA , * * * p ≤ 0 . 001 ) .

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(C) Loss of CHD1 leads to increased sensitivity to PARP inhibition. Control or CHD1-depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks. Data represented as mean values (n=3, ± SD, p-value=0.0003, ANOVA, ***p ≤ 0.001).


( D ) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation . shCont - or shCHD1 - expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X - rays and surviving fractions ( normalized to the unirradiated condition ) were measured by counting colonies after 3 weeks . Data is represented as mean ± SD ( n = 3 , p - value = 0 . 07 , ANOVA , * p ≤ 0 . 1 ) .

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(D) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation. shCont- or shCHD1-expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X-rays and surviving fractions (normalized to the unirradiated condition) were measured by counting colonies after 3 weeks. Data is represented as mean ± SD (n=3, p-value=0.07, ANOVA, *p ≤ 0.1).


( E ) Frequency of CHD1 gene alterations in comparison with DSB repair genes in prostate cancer patients . Data obtained from cBioPortal for Cancer Genomics . ( F ) Frequency of CHD1 gene alterations from indicated data sets . See also Figure EV5A - EV5B .

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(E) Frequency of CHD1 gene alterations in comparison with DSB repair genes in prostate cancer patients. Data obtained from cBioPortal for Cancer Genomics. (F) Frequency of CHD1 gene alterations from indicated data sets. See also Figure EV5A-EV5B.


Blistered samples at P1 . ( B ) Hematoxylin and eosin ( H & E , top ) and alkaline phosphatase ( AP , bottom ) staining . Hair follicles ( HFs ) remaining in the dermis ( indicated by arrows ) . Scale bar : 500 μm . ( C ) α6 Integrin ( ITGA6 , indicated by arrowheads ) and type IV collagen ( COL4 , arrows ) labeling ( left ) . Laminin 332 ( L332 , arrows ) staining ( right ) . Scale bar : 100 μm . ( D ) Ultrastructural findings of blistered skin ( left image : blister roof , right image : blister bottom ) . Hemidesmosomes ( white arrowheads ) and lamina densa ( arrows ) are indicated . Scale bar : 1 μm .

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Blistered samples at P1. (B) Hematoxylin and eosin (H&E, top) and alkaline phosphatase (AP, bottom) staining. Hair follicles (HFs) remaining in the dermis (indicated by arrows). Scale bar: 500 μm. (C) α6 Integrin (ITGA6, indicated by arrowheads) and type IV collagen (COL4, arrows) labeling (left). Laminin 332 (L332, arrows) staining (right). Scale bar: 100 μm. (D) Ultrastructural findings of blistered skin (left image: blister roof, right image: blister bottom) . Hemidesmosomes (white arrowheads) and lamina densa (arrows) are indicated. Scale bar: 1 μm.


Keratin 14 ( K14 ) and keratin 10 ( K10 ) staining of the nonlesional ( intact ) and lesional ( blistered ) skin at P2 ( upper images and inlets : sections , lower images : whole - mount imaging ) . HFs are indicated by arrows in the whole - mount images . Scale bar : 30 μm .

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Keratin 14 (K14) and keratin 10 (K10) staining of the nonlesional (intact) and lesional (blistered) skin at P2 (upper images and inlets: sections, lower images: whole-mount imaging). HFs are indicated by arrows in the whole-mount images. Scale bar: 30 μm.


H & E ( left ) , PCK ( middle ) , and LOR staining ( right ) at P4 . Scale bar : 200 μm .

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H&E (left), PCK (middle), and LOR staining (right) at P4. Scale bar: 200 μm.


Scatter plots of differentially expressed genes in the regenerated epidermis . The red dots represent upregulated genes , and the blue dots represent downregulated genes . The gray dotted lines indicate | logFC | > 1 .

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Scatter plots of differentially expressed genes in the regenerated epidermis. The red dots represent upregulated genes, and the blue dots represent downregulated genes. The gray dotted lines indicate |logFC|>1.


Hair canals in the regenerated ( lesional ) and nonlesional epidermis at P4 ( indicated by asterisks ) . Scale bar : 300 μm .

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Hair canals in the regenerated (lesional) and nonlesional epidermis at P4 (indicated by asterisks). Scale bar: 300 μm.


BrdU labeling of blistered samples at P2 . Scale bar : 100 μm . BrdU - positive cells are indicated by arrows . Blisters are indicated by stars . ( Bottom ) Quantification of BrdU - positive cells in the epidermis ( left ) and HFs ( right ) ( n = 4 biological replicates ) . The data are shown as the mean ± SE . * 0 . 01 test , followed by Tukey ' s test . NS , no significance .

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BrdU labeling of blistered samples at P2. Scale bar: 100 μm. BrdU-positive cells are indicated by arrows. Blisters are indicated by stars. (Bottom) Quantification of BrdU-positive cells in the epidermis (left) and HFs (right) (n=4 biological replicates). The data are shown as the mean ± SE. *0.01 test, followed by Tukey's test. NS, no significance.


α5 integrin ( ITGA5 ) labeling at P2 ( left image : section , right image : whole - mount ) . Scale bar : 100 μm . Blister edges ( epidermal tongue ) and HFs are indicated by arrowheads and arrows , respectively . Blisters are indicated by stars .

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α5 integrin (ITGA5) labeling at P2 (left image: section, right image: whole-mount). Scale bar: 100 μm. Blister edges (epidermal tongue) and HFs are indicated by arrowheads and arrows, respectively. Blisters are indicated by stars.


Sections of K14CreER : H2B - mCherry mice at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . . The data from individual mice are connected by lines . Student ' s t - test . NS , no significance . Whole - mount imaging of K14CreER : R26R - confetti samples at P4 . Scale bar : 200 μm . Sections of Lrig1CreER : H2B - mCherry mouse skin at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . The data from individual mice are connected by lines . * 0 . 01 test . Whole - mount imaging of Lrig1CreER : R26R - confetti mouse samples at P4 . Scale bar : 200 μm .

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Sections of K14CreER:H2B-mCherry mice at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3).. The data from individual mice are connected by lines. Student's t-test. NS, no significance. Whole-mount imaging of K14CreER:R26R-confetti samples at P4. Scale bar: 200 μm. Sections of Lrig1CreER:H2B-mCherry mouse skin at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3). The data from individual mice are connected by lines. *0.01 test. Whole-mount imaging of Lrig1CreER:R26R-confetti mouse samples at P4. Scale bar: 200 μm.


H & E ( P2 , left ) , ITGA6 ( P2 , middle ) and LOR staining ( P4 , right ) of Col17a1 - / - mice ( top ) and littermate controls ( bottom ) . The regenerated epidermis is indicated by arrowheads . Scale bar : 200 μm .

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H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.


BrdU labeling of Col17a1 - / - skin at P2 . Scale bar : 100 μm . ( Bottom ) Quantification of BrdU - positive cells in the epidermis surrounding blisters ( n = 3 ( control ) and 4 ( Col17a1 - / - ) biological replicates ) . The data are shown as the mean ± SE . Student ' s t - test . NS , no significance .

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BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.


Lineage tracing of K14CreER : R26R - mCherry : Col17a1 - / - at P4 . Scale bar : 100 μm .

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Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm.


K10 / K14 ( low magnification , left ) and K14 ( high magnification , middle and right ) labeling of Col7a1 - / - mouse ( bottom ) and littermate control ( top ) blistered skin at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 55 HFs from three control and 143 HFs from four Col7a1 - / - mice ) . The data are shown as violin plots . Student ' s t - test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 244 ( control , L ) , and 132 ( Col7a1 - / - , L ) cells from four mice , respectively ) and in the surrounding intact epidermis ( basal cells ; n = 200 ( control , NL ) , 299 ( Col7a1 - / - , NL ) , from four mice , respectively ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

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K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


K10 / K14 ( left ) and K14 ( high magnification , right ) labeling of WT blistered skin treated with CaCl2 ( middle and bottom ) or PBS ( top ) at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 83 ( PBS ) , 95 ( 1 . 8 mM CaCl2 ) , and 97 ( 9 . 0 mM CaCl2 ) HFs from four mice ) . One - way ANOVA test , followed by Tukey ' s test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 433 ( PBS , L ) , 451 ( 1 . 8 mM CaCl2 , L ) , and 425 ( 9 . 0 mM CaCl2 , L ) cells from four mice ) and in the surrounding intact epidermis ( basal cells ; n = 311 ( PBS , NL ) , 279 ( 1 . 8 mM CaCl2 , NL ) , 302 ( 9 . 0 mM CaCl2 , NL ) cells from four mice ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

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K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


C Overabundance of MIAA in LAM plasma . Top panel shows each control and patient group ( number ( n ) of samples are indicated ) ; bottom panel shows aggregation of related pulmonary diseases . The asterisks indicate significant differences based on two - sided Mann - Whitney tests ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; top panel , control - LAM P = 0 . 048 , LAM - Langerhans P = 1x10 - 4 , LAM - Sjögren P = 1x10 - 4 , LAM - lupus P = 1x10 - 3 , LAM - emphysema P = 0 . 041 ; bottom panel , LAM - Langerhans P = 7x10 - 6 and control - other P = 0 . 031 ) . Average values are indicated with lilac - colored lines . Comparison of the three groups : Kruskal - Wallis test P = 6x10 - 4 ( top panel ) and P = 3x10 - 5 ( bottom panel ) .

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C Overabundance of MIAA in LAM plasma. Top panel shows each control and patient group (number (n) of samples are indicated); bottom panel shows aggregation of related pulmonary diseases. The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; top panel, control-LAM P = 0.048, LAM-Langerhans P = 1x10-4, LAM-Sjögren P = 1x10-4, LAM-lupus P = 1x10-3, LAM-emphysema P = 0.041; bottom panel, LAM-Langerhans P = 7x10-6 and control-other P = 0.031). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test P = 6x10-4 (top panel) and P = 3x10-5 (bottom panel).


E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin - treated ( number ( n ) of samples are indicated ) . The asterisk indicates significant difference based on two - sided Mann - Whitney test ( P = 0 . 043 ) . Average values are indicated with lilac - colored lines .

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E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin-treated (number (n) of samples are indicated). The asterisk indicates significant difference based on two-sided Mann-Whitney test (P = 0.043). Average values are indicated with lilac-colored lines.


G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and / or between LAM and related pulmonary diseases ( individual samples indicated in panel C ) . The asterisks indicate significant differences based on two - sided Mann - Whitney tests ( * P < 0 . 05 , * * P < 0 . 01 , and * * * * P < 0 . 0001 ; 4 - HPA , control - LAM P = 0 . 024 and LAM - other P = 0 . 089 ; HVA , control - LAM P = 0 . 008 and LAM - other P = 0 . 013 ; and VMA , control - LAM P = 1x10 - 4 and LAM - other P = 0 . 10 ) . Average values are indicated with lilac - colored lines . Comparison of the three groups : Kruskal - Wallis test ; 4 - HPA P = 0 . 095 ; HVA P = 0 . 013 ; and VMA P = 0 . 005 .

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G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and/or between LAM and related pulmonary diseases (individual samples indicated in panel C). The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, and ****P < 0.0001; 4-HPA, control-LAM P = 0.024 and LAM-other P = 0.089; HVA, control-LAM P = 0.008 and LAM-other P = 0.013; and VMA, control-LAM P = 1x10-4 and LAM-other P = 0.10). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test; 4-HPA P = 0.095; HVA P = 0.013; and VMA P = 0.005.


A Overabundance of MIAA in LAM plasma ( UK cohort not treated with rapamycin ) relative to healthy women . The number ( n ) of individuals in each group is indicated ; one of 20 healthy controls did not pass the quality controls for LC - MS / MS . Asterisk indicates significance using two - sided Mann - Whitney test ( P = 0 . 018 ) . Average values are indicated with lilac - colored lines . B Overabundance of MIAA in plasma of LAM patients with AMLs . Asterisk indicates significance using two - sided Mann - Whitney test ( P = 0 . 042 ) . The number ( n ) of individuals in each group is indicated . Average values are indicated with lilac - colored lines .

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A Overabundance of MIAA in LAM plasma (UK cohort not treated with rapamycin) relative to healthy women. The number (n) of individuals in each group is indicated; one of 20 healthy controls did not pass the quality controls for LC-MS/MS. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.018). Average values are indicated with lilac-colored lines. B Overabundance of MIAA in plasma of LAM patients with AMLs. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.042). The number (n) of individuals in each group is indicated. Average values are indicated with lilac-colored lines.


A Representative images of immunohistochemical positivity of MAO - A / B in LAM lung lesions ( top panels , two patients ) and lung tissue from healthy ( non - LAM ) individuals ( bottom panels ) . In total , seven LAM patients and three healthy controls were analyzed . Brown - stained cells , counter - stained with hematoxylin , are considered positive . Scale bars are shown .

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A Representative images of immunohistochemical positivity of MAO-A/B in LAM lung lesions (top panels, two patients) and lung tissue from healthy (non-LAM) individuals (bottom panels). In total, seven LAM patients and three healthy controls were analyzed. Brown-stained cells, counter-stained with hematoxylin, are considered positive. Scale bars are shown.


B Representative images of immunofluorescence detection of MAO - A / B and αSMA in LAM lung lesions , nuclei stained with DAPI ( merged ) .

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B Representative images of immunofluorescence detection of MAO-A/B and αSMA in LAM lung lesions, nuclei stained with DAPI (merged).


C Graph showing gene expression differences ( log10 - fold changes ) of defined genes ( X - axis ) in Tsc2 - deficient relative to wild - type MEFs grown in DMEM 10 % FBS . The asterisks indicate significant differences with two - sided t - test ( * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; Aldh1a3 P = 0 . 006 , Aldh2 P = 2x10 - 5 , Aldh3a1 P = 0 . 004 ; Aldh3b1 P = 0 . 006 ; Aldh3b2 , P = 0 . 17 , Maoa P = 7x10 - 5 , and Maob P = 7x10 - 4 ; replicates / condition n = 3 , assays n = 4 ) . The bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) and 0 . 5 - fold ( bottom ) .

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C Graph showing gene expression differences (log10-fold changes) of defined genes (X-axis) in Tsc2-deficient relative to wild-type MEFs grown in DMEM 10% FBS. The asterisks indicate significant differences with two-sided t-test (**P < 0.01, ***P < 0.001, and ****P < 0.0001; Aldh1a3 P = 0.006, Aldh2 P = 2x10-5, Aldh3a1 P = 0.004; Aldh3b1 P = 0.006; Aldh3b2, P = 0.17, Maoa P = 7x10-5, and Maob P = 7x10-4; replicates/condition n = 3, assays n = 4). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).


D Western blot results ( independent experiments n = 4 ) from Tsc2 - deficient and wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % FBS . Loading controls are shown . The inferred protein expression level is indicated by the ratio between the corresponding signal and loading control , standardized to the basal

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D Western blot results (independent experiments n = 4) from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% FBS. Loading controls are shown. The inferred protein expression level is indicated by the ratio between the corresponding signal and loading control, standardized to the basal setting


A No differences of Vdac1 / VDAC1 expression between MEF cell lines ( replicates / condition n = 3 and independent experiments n = 2 ) . The graph shows fold change expression ( mean ± SEM ) in Tsc2 - deficient relative to wild - type MEFs .

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A No differences of Vdac1/VDAC1 expression between MEF cell lines (replicates/condition n = 3 and independent experiments n = 2). The graph shows fold change expression (mean ± SEM) in Tsc2-deficient relative to wild-type MEFs.


B Flow cytometry results ( independent experiments n = 3 ) showing a higher percentage of MitoSOX red - positive cells in Tsc2 - deficient MEFs . C Flow cytometry results ( independent experiments n = 3 ) showing higher intensity ( X - axis , single channel intensity FL3 670 nm ) of MitoTracker red - positive cells in Tsc2 - deficient MEFs .

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B Flow cytometry results (independent experiments n = 3) showing a higher percentage of MitoSOX red-positive cells in Tsc2-deficient MEFs. C Flow cytometry results (independent experiments n = 3) showing higher intensity (X-axis, single channel intensity FL3 670 nm) of MitoTracker red-positive cells in Tsc2-deficient MEFs.


D Higher basal cell respiration ( as measured by oxygen consumption , Y - axis ) in Tsc2 - deficient MEFs . The asterisks indicate significant difference with two - sided t - test ( P = 5x10 - 4 ; replicates / condition n = 5 , and independent experiments n = 3 ) .

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D Higher basal cell respiration (as measured by oxygen consumption, Y-axis) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 5x10-4; replicates/condition n = 5, and independent experiments n = 3).


F Left panel , western blot results showing overexpression of catalase ( CAT ) in Tsc2 - deficient MEFs , and unaffected by exposure to everolimus ( independent experiments n = 2 ) . The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting ( noted as 1 ( ref ) ) . Right panel , Cat overexpression ( log10 - fold change ) in Tsc2 - deficient MEFs . The asterisks indicate significant difference with two - sided t - test ( P = 1x10 - 2 ; replicates / condition n = 3 , and independent experiments n = 2 ) . The bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) and 0 . 5 - fold ( bottom ) .

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F Left panel, western blot results showing overexpression of catalase (CAT) in Tsc2-deficient MEFs, and unaffected by exposure to everolimus (independent experiments n = 2). The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting (noted as 1(ref)). Right panel, Cat overexpression (log10-fold change) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 1x10-2; replicates/condition n = 3, and independent experiments n = 2). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).


G Representative images of immunohistochemical detection of acrolein in LAM lung lesions .

G Representative images of immunohistochemical detection of acrolein in LAM lung lesions.


A Overabundance of histamine in Tsc2 wild - type relative to Tsc2 - deficient MEFs , both growth in DMEM 10 % FBS . The asterisks indicate significant difference with two - sided t - test ( P = 0 . 017 ) ; replicates / condition n = 5 , and independent experiments n = 2 ) . Average values are indicated with lilac - colored lines .

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A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines.


D Western blot results of HRH1 , SLC22A3 , and loading control from Tsc2 - deficient and wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % or 0 . 5 % FBS ( independent experiments n = 2 ) . The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal

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D Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal setting


E Representative images of immunohistochemical detection of HRH1 in LAM lung lesions .

E Representative images of immunohistochemical detection of HRH1 in LAM lung lesions.


F Evaluation of cell viability inhibition by loratadine alone ( concentrations shown on X - axis , from 0 to 100 μM ) or combined with everolimus ( fixed to 20 nM ) in Tsc2 - deficient and wild - type MEF cultures grown in DMEM 10 % FBS for 72 hours . The synergistic combination index ( CIx < 1 ) is shown ( replicates / condition n = 3 , independent experiments n = 3 ) . Each data point represents the mean and SD .

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F Evaluation of cell viability inhibition by loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD.


H Percentages of viability of Tsc2 - dificient and wild - type MEFs exposed to DMEM 10 % or 0 . 5 % FBS with or without L - histidine for 72 hours . The asterisks indicate significant differences with one - sided t - test ( 10 % FBS , P = 0 . 017 ; 0 . 5 % FBS , P = 5x10 - 4 ; replicates / condition n = 4 - 6 , independent experiments n = 2 ) . The bars indicate mean ± SD .

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H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5% FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10% FBS, P = 0.017; 0.5% FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.


B Inhibition of Tsc2 - deficient 105K tumor growth with different monotherapies , as indicated in the inset . Asterisks indicate significant reductions relative to vehicle ( two - way ANOVA ; clorgyline P = 0 . 015 , loratadine P = 0 . 018 , and rasagiline P = 0 . 049 ) . Each data point represents the mean and SEM .

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B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM.


C Further reduction of Tsc2 - deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine , relative to rapamycin alone . Asterisks indicate significant reductions relative to rapamycin alone ( two - way ANOVA ; clorgyline P = 0 . 035 , loratadine P = 0 . 045 ) . Each data point represents the mean and SEM .

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C Further reduction of Tsc2-deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM.


E Inhibition of Tsc2 - deficient 105K tumor growth with Maoa expression depletion using shRNAs . Asterisks indicate significant reductions relative to control pLKO ( two - way ANOVA ; P = 1x10 - 3 ) . Each data point represents the mean and SEM .

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E Inhibition of Tsc2-deficient 105K tumor growth with Maoa expression depletion using shRNAs. Asterisks indicate significant reductions relative to control pLKO (two-way ANOVA; P = 1x10-3). Each data point represents the mean and SEM.


F Inhibition of Tsc2 - deficient 105K tumor growth with Hrh1 expression depletion . Asterisk indicates significant reduction relative to control pLKO ( two - way ANOVA ; P = 0 . 035 ) . Each data point represents the mean and SEM . G Inhibition of Tsc2 - deficient 105K tumor growth with administration of α - methyl - DL - histidine . Asterisk indicates significant reduction relative to vehicle ( two - way ANOVA ; P = 0 . 028 ) . Each data point represents the mean and SEM .

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F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM. G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM.


A Top panels , western blot results of HRH1 and phospho - Ser616 DRP1 expression in vehicle control and single - drug treated tumors . Bottom panels show quantifications ; significant differences correspond to one - sided t - test ( * P = 0 . 011 and * * P = 2x10 - 3 ; tumors / group n = 4 ) . Average values are indicated with lilac - colored lines . B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone , reduced expression of S6 total in the rapamycin - loratadine combination , and raised phospho - Ser616 DRP1 expression in this combination . Bottom panels show quantifications and significant differences with one - sided t - test ( HRH1 , clorgyline P = 0 . 011 , loratadine P = 0 . 049 , rasagiline P = 0 . 021 ; total S6 , loratadine P = 4x10 - 4 ; phospho - Ser616 DRP1 , loratadine P = 0 . 037 ; tumors / group n = 4 ) . Average values are indicated with lilac - colored lines .

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A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1 expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines. B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1, loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines.


E Representative images of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with vehicle or monotherapies . Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis , whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology . Scale bars are shown .

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E Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown.


F Representative images of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with rapamycin alone or rapamycin combinations . With the addition of a second drug to rapamycin , tumors more frequently tended to show glandular differentiation and less atypia . Scale bars are shown .

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F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.


( B ) Violin plots showing the expression of prostaglandin ( PG ) receptors in pre - adipocytes and mature adipocytes from human deep - neck BAT ( E - MTAB - 8564 ) .

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(B) Violin plots showing the expression of prostaglandin (PG) receptors in pre-adipocytes and mature adipocytes from human deep-neck BAT(E-MTAB-8564).


( A ) Oil Red O staining of mouse BAs differentiated from normal or EP3 - deficient brown pre - adipocytes . Scale bar : 50 μm . ( B ) Mean lipid droplet area of differentiated BAs from normal or EP3 - deficient brown pre - adipocytes . * P < 0 . 05 vs WT ( n = 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U tests ( B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 .

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(A) Oil Red O staining of mouse BAs differentiated from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm. (B) Mean lipid droplet area of differentiated BAs from normal or EP3-deficient brown pre-adipocytes. *P < 0.05 vs WT (n = 6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests (B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05


( D ) Representative image of hematoxylin and eosin ( H & E ) staining for iBAT from EP3F / F and EP3F / FMyf5Cre mice . Scale bar : 50 μm . ( E ) Mean adipocyte area of BAs in iBAT from EP3F / F and EP3F / FMyf5Cre mice ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U tests E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(D) Representative image of hematoxylin and eosin (H&E) staining for iBAT from EP3F/F and EP3F/FMyf5Cre mice. Scale bar: 50 μm. (E) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( K ) Representative image of H & E staining of iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD . Scale bar : 50 μm . ( L ) Mean adipocyte area of BAs in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are pooled from two independent experiments with biological replicates L , Statistical significance was evaluated by Mann - Whitney U tests L , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(K) Representative image of H&E staining of iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD. Scale bar: 50 μm. (L) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates L, Statistical significance was evaluated by Mann-Whitney U tests L, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( A ) N6 - methyladenosine ( m6A ) modification levels of mRNAs in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction , detected by m6A RNA Methylation Quantification Kit ( Colorimetric ) ( n = 4 - 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test ( A * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(A) N6-methyladenosine (m6A) modification levels of mRNAs in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction, detected by m6A RNA Methylation Quantification Kit (Colorimetric) (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test (A *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( B ) qRT - PCR analysis of the relative mRNA levels of m6A writers ( METTL3 , METTL4 , METTL14 and WTAP ) and erasers ( FTO and ALKBH5 ) in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction ( n = 6 - 9 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(B) qRT-PCR analysis of the relative mRNA levels of m6A writers (METTL3, METTL4, METTL14 and WTAP) and erasers (FTO and ALKBH5) in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction (n = 6-9). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( C ) Western blotting analysis of m6A writers and erasers in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction .

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(C) Western blotting analysis of m6A writers and erasers in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction.


( D ) Quantification of protein levels in C ( n = 4 - 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates , D , Statistical significance was evaluated by unpaired two - tailed student ' s t test D , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(D) Quantification of protein levels in C (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates , D, Statistical significance was evaluated by unpaired two-tailed student's t test D, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( E ) Western blotting analysis of WTAP in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD for 1 week .

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(E) Western blotting analysis of WTAP in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD for 1 week.


( F ) Western blotting analysis of WTAP in BAs differentiated from EP3γ - expressed brown pre - adipocytes .

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(F) Western blotting analysis of WTAP in BAs differentiated from EP3γ-expressed brown pre-adipocytes.


( G ) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ - expressed brown pre - adipocytes detected by RT - qPCR ( n = 6 - 7 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates G , Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons ( G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(G) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed brown pre-adipocytes detected by RT-qPCR (n = 6-7). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates G, Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons (G, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( H ) qRT - PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . ( I ) qRT - PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test H ) , two - way ANOVA followed by Tukey ' s test for post - hoc comparisons I , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(H) qRT-PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). (I) qRT-PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test H), two-way ANOVA followed by Tukey's test for post-hoc comparisons I, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( J ) Representative picture of oil red O - stained BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes with WTAP siRNA treatment . Scale bar : 50 μm .

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(J) Representative picture of oil red O-stained BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes with WTAP siRNA treatment. Scale bar: 50 μm.


( K - Q ) qRT - PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons K - Q ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(K-Q) qRT-PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons K-Q). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( A ) Western blotting analysis of ubiquitination of WTAP in the control and EP3 - deficient brown pre - adipocytes with or without sulprostone treatment . Data information : Data are representative of two independent experiments with biological

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(A) Western blotting analysis of ubiquitination of WTAP in the control and EP3-deficient brown pre-adipocytes with or without sulprostone treatment. Data information: Data are representative of two independent experiments with biological replicates


( B ) Western blotting analysis of ERK1 / 2 phosphorylation in the control and EP3 - deficient brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(B) Western blotting analysis of ERK1/2 phosphorylation in the control and EP3-deficient brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( C ) Effect of ERK1 / 2 inhibitor , ravoxertinib , on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(C) Effect of ERK1/2 inhibitor, ravoxertinib, on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( D ) Effect of wortmannin ( Wor ) , pertussis toxin ( PTX ) , U73122 , barbadin , and melittin on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(D) Effect of wortmannin (Wor), pertussis toxin (PTX), U73122, barbadin, and melittin on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( E ) Analysis of PKA activity in the control and EP3 - deficient brown pre - adipocytes ( n = 4 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological

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(E) Analysis of PKA activity in the control and EP3-deficient brown pre-adipocytes (n = 4). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates


( F ) Effect of PKA inhibitor , H89 , on ubiquitin - dependent degradation of WTAP in EP3 - / - brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(F) Effect of PKA inhibitor, H89, on ubiquitin-dependent degradation of WTAP in EP3-/- brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( G ) Effect of ERK1 / 2 phosphorylation site mutation ( HA - WTAP - 2A ) on EP3 - mediated ubiquitin - dependent degradation of WTAP in brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(G) Effect of ERK1/2 phosphorylation site mutation (HA-WTAP-2A) on EP3-mediated ubiquitin-dependent degradation of WTAP in brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( H ) Representative picture of oil red O - stained BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological

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(H) Representative picture of oil red O-stained BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates


( I ) Western blotting assay of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , and UCP1 in BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Data information : Data are representative of two independent experiments with biological

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(I) Western blotting assay of ERK1/2, WTAP, ZFP410, PRDM16, and UCP1 in BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Data information: Data are representative of two independent experiments with biological replicates


( B ) Representative pictures of nile red - stained human BAs differentiated from hESC - derived brown progenitors . Scale bar : 50 μm .

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(B)Representative pictures of nile red-stained human BAs differentiated from hESC-derived brown progenitors. Scale bar: 50 μm.


( C ) Time course analysis of the relative mRNA levels of EP3 receptors in hESC - derived BAs during BA differentiation detected by RT - qPCR ( n = 7 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates ( C , Statistical significance was evaluated by Welch ANOVA test followed by Tamhane ' s T2 test for post - hoc comparisons ( C ) * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(C) Time course analysis of the relative mRNA levels of EP3 receptors in hESC-derived BAs during BA differentiation detected by RT-qPCR (n = 7-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates (C, Statistical significance was evaluated by Welch ANOVA test followed by Tamhane's T2 test for post-hoc comparisons (C) *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( D ) Representative pictures of hESC - derived BAs treated with L798106 alone or combined with H89 . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological

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(D) Representative pictures of hESC-derived BAs treated with L798106 alone or combined with H89. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates


( E ) Effect of H89 on the relative mRNA level of brown marker gene in hESC - derived BAs treated with L798106 detected by RT - qPCR ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for multiple comparisons ( E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(E) Effect of H89 on the relative mRNA level of brown marker gene in hESC-derived BAs treated with L798106 detected by RT-qPCR (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons (E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( F ) Effect of H89 on WTAP mRNA expression in hESC - derived BAs treated with L798106 ( n = 6 - 7 ) . ( G ) Effect of H89 on ZNF410 mRNA expression in hESC - derived BAs treated with L798106 ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two - way ANOVA followed by Tukey ' s test for multiple comparisons F , G ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(F) Effect of H89 on WTAP mRNA expression in hESC-derived BAs treated with L798106 (n = 6-7). (G) Effect of H89 on ZNF410 mRNA expression in hESC-derived BAs treated with L798106 (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two-way ANOVA followed by Tukey's test for multiple comparisons F, G). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( H ) Western blotting assay of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , and UCP1 in hESC - derived BAs treated with L798106 alone or combined with H89 .

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(H) Western blotting assay of ERK1/2, WTAP, ZNF410, PRDM16, and UCP1 in hESC-derived BAs treated with L798106 alone or combined with H89.


( A ) Comparison of migration and invasion abilities between T - and B - lines . PC3 T - and B - line cells were incubated for migration ( 16h ) and invasion ( 24h ) assay . Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ) .

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(A) Comparison of migration and invasion abilities between T- and B- lines. PC3 T- and B-line cells were incubated for migration (16h) and invasion (24h) assay. Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).


( B ) Comparison of cell proliferation rate between T - and B - lines . Cell proliferation assay was performed by MTS reagents . Data are the means ± SD ( n = 3 biological replicates ) .

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(B) Comparison of cell proliferation rate between T- and B- lines. Cell proliferation assay was performed by MTS reagents. Data are the means ± SD (n=3 biological replicates).


( C ) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor - derived ( T2 and T3 - lines ) and bone - metastases - derived cancer cells ( B2 and B3 - lines ) .

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(C) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor-derived (T2 and T3-lines) and bone-metastases-derived cancer cells (B2 and B3-lines).


( D ) Top : H2S catalyzing activity of cell lysates from PC3 T2 , B2 , T3 , and B3 lines . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : The level of H2S production was quantified by densitometry , and the histograms represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis ( * P < 0 . 05 ) .

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(D) Top: H2S catalyzing activity of cell lysates from PC3 T2, B2, T3, and B3 lines. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).


( E , F ) 1x106 PC3 - T2 , B2 , T3 , and B3 cells were orthotopically injected into mouse prostate for 15 - 20 days . Incidences of paraaortic lymph nodes metastasis ( E ) and bone metastasis ( F ) are shown ( n = 6 - 8 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ) .

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(E, F) 1x106 PC3-T2, B2, T3, and B3 cells were orthotopically injected into mouse prostate for 15-20 days. Incidences of paraaortic lymph nodes metastasis (E) and bone metastasis (F) are shown (n=6-8 mice per group). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).


( G ) Top : expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays . Samples were sub - grouped by TNM stages . The representative images from different stages are shown . S : Stroma ; Scale bars : 250μm . Bottom : The statistical significance was determined using the χ2

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(G) Top: expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays. Samples were sub-grouped by TNM stages. The representative images from different stages are shown. S: Stroma; Scale bars: 250μm. Bottom: The statistical significance was determined using the χ2 test


The RNA - seq data of CTH was downloaded from the TCGA database ( H ) The mRNA expression of CTH in PC specimens and normal tissues . Data are presented as means ± SD ( n = 50 cases for adjancent non - tumor parts and 469 cases for PC tumors ) . Student ' s t - test was used for the statistical analysis ( * * P < 0 . 01 ) .

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The RNA-seq data of CTH was downloaded from the TCGA database (H) The mRNA expression of CTH in PC specimens and normal tissues. Data are presented as means ± SD (n=50 cases for adjancent non-tumor parts and 469 cases for PC tumors). Student's t-test was used for the statistical analysis (**P<0.01).


( I ) Kaplan - Meier survival analysis of PC patients with high or low CTH expression . The statistical significance was determined using the χ2 test .

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(I) Kaplan-Meier survival analysis of PC patients with high or low CTH expression. The statistical significance was determined using the χ2 test.