( D ) CHD1 - depleted show increased sensitivity to γ - radiation . For colony formation , both BHP1 PC3 with shCont or shCHD1 were treated with indicated doses of γ - radiation surviving fractions were measured by counting colonies after 3 , mean values are represented in plot ( n = 3 , ± SD ) . Data were normalized to plating efficiency . p - values ( 0 . 0009 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 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 with I - Sce - I - expressing vector ( pCMV - I - SceI - 3xNLS ) . After 48 h of transfection , GFP - positive were measured by flow cytometry . HR ( A ) or NHEJ ( B ) efficiency was calculated on fraction of GFP - positive represented as mean values from three independent experiments as ± SD , 50 , 000 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 show decreased RAD51 foci after damage induction . shCont or shCHD1 PC3 were irradiated co - immunostained with γH2AX RAD51 antibodies after indicated time points . Scale bar 5 µm . ( D ) number of RAD51 foci co - localized with γH2AX per ( from C ) were counted ( n = 50 ) represented in a graph . data is represented in mean value of three independent experiments as ± SD , more than 50 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 with CtIP CHD1 in PC3 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 shCHD1 PC3 were treated with NCS for indicated times fractions were immunoblotted for CtIP , RPA1 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 ( D ) RPA1 recruitment to I - SceI - induced DSB in U2OS19 ptight13 GFP - LacR . CHD1 was depleted in U2OS19 ptight13 GFP - LacR by siRNA 48 h after transfection were treated with doxy for 16 h co - immunostained for γH2AX CtIP or RPA1 . number of with CtIP or RPA1 foci from ( C ) ( D ) were counted represented mean in graph as of foci positive as ± SD ( n = 3 ) , more than 50 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 were transfected either with mock or siCHD1 followed by empty vector , wt mChd1 ( Wt ) or ATPase - mutant ( Mt ) mChd1 . After 48 h of transfection were treated with of 4 - OH tamoxifen ( 4 - OHT ) for 24 h processed for PLA with γH2AX CtIP antibodies . demonstrating positive focal interactions indicative of repair hubs ( punctate ) are present only in control ( Wt ) CHD1 - rescued 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 which stably express HA - mChd1 - ERT2 , were transfected with either mock or siCHD1 . After 24 h of transfection were treated with 4 - OHT for 24 h to induce HA - mChd1 - ERT2 translocation . Western blot of fractions for CHD1 , ERT2 ( mChd1 ) , CtIP RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ . relative for indicated is shown in 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 were transfected with mock or siMRE11 ( SmartPool ) 48 h after transfection were treated with NCS fractions were isolated 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 CHD1 antibodies in mock or CtIP depleted after 2 h of NCS . Scale bar is 50 µm . Verification of knockdown efficiency is shown by Western blot on right . HSC70 is shown as a loading control . Scale bar 20 µm . ( C ) of PLA signal from ( B ) represented as mean values from three independent experiments as ± SD , more than 100 were counted per condition .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 accessibility at two HR - repaired sites ( DSB - I DSB - II ) was analyzed by FAIRE in AsiSI - ER - U20S transfected with either mock or CHD1 siRNA . After 48 h of transfection were treated with 4 - OHT for indicated time points processed for FAIRE . data are represented as mean ± SD ( n = 3 ) p - values were calculated using ANOVA ( 0 . 02 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 of PC3 transfected either with mock or siCHD1 ( SmartPool ) were grown for 48 h then treated with NCS for 2 h prior to with anti - BrdU 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 ) of Brdu positive from ( E ) . data are represented as mean ± SD from three independent experiments , more than 50 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 show hypersensitivity to Mitomycin ( MMC ) treatment . For colony formation shCont shCHD1 PC3 were treated with indicated MMC concentrations for 4 h surviving fractions were measured by counting colonies after 3 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 show increased hypersensitivity to Irinotecan . For proliferation , shCont shCHD1 were treated with 1 µM Irinotecan proliferation was measured by Celigo relative confluency ( mean ) was plotted in 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 were treated with indicated concentrations of PARP inhibitor olaparib surviving fractions were measured by counting colonies after 3 . 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 sensitive to PARP inhibition in combination with irradiation . shCont - or shCHD1 - expressing BHP1 were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X - rays surviving fractions ( normalized to unirradiated condition ) were measured by counting colonies after 3 . 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 . 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 eosin ( H & E , top ) alkaline phosphatase ( AP , bottom ) . Hair follicles ( HFs ) remaining in dermis ( indicated by arrows ) . Scale bar : 500 μm . ( C ) α6 Integrin ( ITGA6 , indicated by arrowheads ) type IV collagen ( COL4 , arrows ) labeling ( left ) . Laminin 332 ( L332 , arrows ) ( right ) . Scale bar : 100 μm . ( D ) Ultrastructural findings of blistered skin ( left image : blister roof , right image : blister bottom ) . Hemidesmosomes ( arrowheads ) 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 ) keratin 10 ( K10 ) of nonlesional ( intact ) lesional ( blistered ) skin at P2 ( upper inlets : sections , lower : whole - mount imaging ) . HFs are indicated by arrows in whole - mount . 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 ) , LOR ( 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 regenerated epidermis . red dots represent upregulated genes , blue dots represent downregulated genes . 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 regenerated ( lesional ) 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 are indicated by arrows . Blisters are indicated by stars . ( Bottom ) of BrdU - positive in epidermis ( left ) HFs ( right ) ( n = 4 biological replicates ) . data are shown as mean ± SE . * 0 . 01 , followed by Tukey ' s . 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 ) HFs are indicated by arrowheads 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 ) of mCherry - positive ( n = 3 ) . . data from mice are connected by lines . Student ' s t - . 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 ) of mCherry - positive ( n = 3 ) . data from mice are connected by lines . * 0 . 01 . 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 ) LOR ( P4 , right ) of Col17a1 - / - mice ( top ) littermate controls ( bottom ) . regenerated epidermis is indicated by arrowheads . 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.


BrdU labeling of Col17a1 - / - skin at P2 . Scale bar : 100 μm . ( Bottom ) of BrdU - positive in epidermis surrounding blisters ( n = 3 ( control ) 4 ( Col17a1 - / - ) biological replicates ) . data are shown as mean ± SE . Student ' s t - . 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 ) K14 ( high magnification , middle right ) labeling of Col7a1 - / - mouse ( bottom ) littermate control ( top ) blistered skin at P2 . Scale bar : 30 μm . of BrdU - positive per μm HF ( n = 55 HFs from three control 143 HFs from four Col7a1 - / - mice ) . data are shown as violin plots . Student ' s t - . NS , no significance . of major axis of keratinocytes in regenerated epidermis ( n = 244 ( control , L ) , 132 ( Col7a1 - / - , L ) from four mice , respectively ) in surrounding intact epidermis ( basal ; n = 200 ( control , NL ) , 299 ( Col7a1 - / - , NL ) , from four mice , respectively ) . NL : nonlesional . L : lesional . data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA , followed by Tukey ' s . 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 ) K14 ( high magnification , right ) labeling of WT blistered skin treated with CaCl2 ( middle bottom ) or PBS ( top ) at P2 . Scale bar : 30 μm . of BrdU - positive per μm HF ( n = 83 ( PBS ) , 95 ( 1 . 8 mM CaCl2 ) , 97 ( 9 . 0 mM CaCl2 ) HFs from four mice ) . One - way ANOVA , followed by Tukey ' s . NS , no significance . of major axis of keratinocytes in regenerated epidermis ( n = 433 ( PBS , L ) , 451 ( 1 . 8 mM CaCl2 , L ) , 425 ( 9 . 0 mM CaCl2 , L ) from four mice ) in surrounding intact epidermis ( basal ; n = 311 ( PBS , NL ) , 279 ( 1 . 8 mM CaCl2 , NL ) , 302 ( 9 . 0 mM CaCl2 , NL ) from four mice ) . NL : nonlesional . L : lesional . data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA , followed by Tukey ' s . NS , no significance .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 group ( number ( n ) of samples are indicated ) ; bottom panel shows aggregation of related pulmonary diseases . asterisks indicate significant differences on two - sided Mann - Whitney ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * 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 control - other P = 0 . 031 ) . Average values are indicated with lilac - colored lines . Comparison of three groups : Kruskal - Wallis P = 6x10 - 4 ( top panel ) P = 3x10 - 5 ( bottom panel ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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 not treated with rapamycin relative to rapamycin - treated ( number ( n ) of samples are indicated ) . asterisk indicates significant difference on two - sided Mann - Whitney ( 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 of three other metabolic products derived from monoamine metabolism showing significant differences from controls / or between LAM related pulmonary diseases ( samples indicated in panel C ) . asterisks indicate significant differences on two - sided Mann - Whitney ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; 4 - HPA , control - LAM P = 0 . 024 LAM - other P = 0 . 089 ; HVA , control - LAM P = 0 . 008 LAM - other P = 0 . 013 ; VMA , control - LAM P = 1x10 - 4 LAM - other P = 0 . 10 ) . Average values are indicated with lilac - colored lines . Comparison of three groups : Kruskal - Wallis ; 4 - HPA P = 0 . 095 ; HVA P = 0 . 013 ; VMA P = 0 . 005 .

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

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

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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 . asterisks indicate significant differences with two - sided t - ( * * P < 0 . 01 , * * * P < 0 . 001 , * * * * 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 , Maob P = 7x10 - 4 ; replicates / condition n = 3 , assays n = 4 ) . bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) 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 wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % FBS . Loading controls are shown . inferred expression level is indicated by between corresponding signal loading control , standardized to basal

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 lines ( replicates / condition n = 3 independent experiments n = 2 ) . graph shows fold change expression ( mean ± ) in Tsc2 - deficient relative to wild - type MEFs .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 respiration ( as measured by oxygen consumption , Y - axis ) in Tsc2 - deficient MEFs . asterisks indicate significant difference with two - sided t - ( P = 5x10 - 4 ; replicates / condition n = 5 , 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 , unaffected by exposure to everolimus ( independent experiments n = 2 ) . CAT expression level is indicated by of corresponding signal relative to loading control basal setting ( noted as 1 ( ref ) ) . Right panel , Cat overexpression ( log10 - fold change ) in Tsc2 - deficient MEFs . asterisks indicate significant difference with two - sided t - ( P = 1x10 - 2 ; replicates / condition n = 3 , independent experiments n = 2 ) . bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) 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 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 . asterisks indicate significant difference with two - sided t - ( P = 0 . 017 ) ; replicates / condition n = 5 , independent experiments n = 2 ) . Average values are indicated with lilac - colored lines .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 , loading control from Tsc2 - deficient 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 ) . expression are indicated by of corresponding signal relative to loading control basal

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of immunohistochemical detection of HRH1 in LAM lung lesions .

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


F Evaluation of 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 wild - type MEF cultures grown in DMEM 10 % FBS for 72 hours . synergistic combination index ( CIx < 1 ) is shown ( replicates / condition n = 3 , independent experiments n = 3 ) . Each data point represents mean 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 wild - type MEFs exposed to DMEM 10 % or 0 . 5 % FBS with or without L - histidine for 72 hours . asterisks indicate significant differences with one - sided t - ( 10 % FBS , P = 0 . 017 ; 0 . 5 % FBS , P = 5x10 - 4 ; replicates / condition n = 4 - 6 , independent experiments n = 2 ) . bars indicate mean ± SD .

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

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 growth with different monotherapies , as indicated in inset . Asterisks indicate significant reductions relative to vehicle ( two - way ANOVA ; clorgyline P = 0 . 015 , loratadine P = 0 . 018 , rasagiline P = 0 . 049 ) . Each data point represents mean .

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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 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 mean .

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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 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 mean .

O O O O O O O O O O O O O O O O O O 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 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 growth with Hrh1 expression depletion . Asterisk indicates significant reduction relative to control pLKO ( two - way ANOVA ; P = 0 . 035 ) . Each data point represents mean . G Inhibition of Tsc2 - deficient 105K 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 mean .

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

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 phospho - Ser616 DRP1 expression in vehicle control single - drug treated tumors . Bottom panels show quantifications ; significant differences correspond to one - sided t - ( * P = 0 . 011 * * 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 rapamycin - loratadine combination , raised phospho - Ser616 DRP1 expression in this combination . Bottom panels show quantifications significant differences with one - sided t - ( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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 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 foci of fibrosis , whereas most of tumors of other treatment groups showed extensive epithelioid . 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 of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with rapamycin alone or rapamycin combinations . With addition of a second drug to rapamycin , tumors more frequently tended to show glandular differentiation 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 expression of prostaglandin ( PG ) receptors in pre - adipocytes 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 of mouse BAs differentiated from normal or EP3 - deficient brown pre - adipocytes . Scale bar : 50 μm . ( B ) Mean lipid droplet of differentiated BAs from normal or EP3 - deficient brown pre - adipocytes . * P < 0 . 05 vs WT ( n = 6 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U ( B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 eosin ( H & E ) for iBAT from EP3F / F EP3F / FMyf5Cre mice . Scale bar : 50 μm . ( E ) Mean adipocyte of BAs in iBAT from EP3F / F EP3F / FMyf5Cre mice ( n = 8 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of iBAT from EP3F / F EP3F / FMyf5Cre mice fed HFD . Scale bar : 50 μm . ( L ) Mean adipocyte of BAs in iBAT from EP3F / F EP3F / FMyf5Cre mice fed HFD ( n = 8 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are pooled from two independent experiments with biological replicates L , Statistical significance was evaluated by Mann - Whitney U L , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of mRNAs in normal EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction , detected by m6A Methylation Kit ( Colorimetric ) ( n = 4 - 6 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - student ' s t ( A * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

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

(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 of relative mRNA of m6A writers ( METTL3 , METTL4 , METTL14 WTAP ) erasers ( FTO ALKBH5 ) in normal EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction ( n = 6 - 9 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - student ' s t B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of m6A writers erasers in normal 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 ) of in C ( n = 4 - 6 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates , D , Statistical significance was evaluated by unpaired two - student ' s t D , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of WTAP in iBAT from EP3F / F 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 of WTAP in BAs differentiated from EP3γ - expressed brown pre - adipocytes .

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


( G ) Relative mRNA of WTAP gene in BAs differentiated from EP3γ - expressed brown pre - adipocytes detected by RT - qPCR ( n = 6 - 7 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates G , Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s for post - hoc comparisons ( G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of relative mRNA of EP3γ gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . ( I ) qRT - PCR of relative mRNA of WTAP gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - student ' s t H ) , two - way ANOVA followed by Tukey ' s for post - hoc comparisons I , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of relative mRNA 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 mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s for post - hoc comparisons K - Q ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of ubiquitination of WTAP in control 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 of ERK1 / 2 phosphorylation in control 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 , 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 ) of PKA activity in control EP3 - deficient brown pre - adipocytes ( n = 4 ) . Data information : Data represent mean ± standard error of mean ( ) . 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 of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , 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 of relative mRNA of EP3 receptors in hESC - derived BAs during BA differentiation detected by RT - qPCR ( n = 7 - 8 ) . Data information : Data represent mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates ( C , Statistical significance was evaluated by Welch ANOVA followed by Tamhane ' s T2 for post - hoc comparisons ( C ) * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ns , not significant ( P > 0 . 05 ) .

(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 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 mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s for multiple comparisons ( E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 mean ± standard error of mean ( ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two - way ANOVA followed by Tukey ' s for multiple comparisons F , G ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 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 of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , 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 invasion abilities between T - B - lines . PC3 T - B - were incubated for migration ( 16h ) invasion ( 24h ) . Data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - was used for statistical ( * 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 proliferation rate between T - B - lines . proliferation was performed by MTS reagents . Data are 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 of endogenous CTH in PC3 orthotopically implanted - derived ( T2 T3 - lines ) bone - metastases - derived cancer ( B2 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 lysates from PC3 T2 , B2 , T3 , B3 lines . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : level of H2S production was quantified by densitometry , histograms represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical ( * 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 , B3 were orthotopically injected into mouse prostate for 15 - 20 days . Incidences of paraaortic lymph nodes metastasis ( E ) bone metastasis ( F ) are shown ( n = 6 - 8 mice per group ) . Student ' s t - was used for statistical ( * 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 of commercial tissue arrays . Samples were sub - grouped by TNM stages . representative from different stages are shown . S : Stroma ; Scale bars : 250μm . Bottom : statistical significance was determined using 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


- seq data of CTH was downloaded from TCGA database ( H ) mRNA expression of CTH in PC specimens normal tissues . Data are presented as means ± SD ( n = 50 cases for adjancent non - parts 469 cases for PC tumors ) . Student ' s t - was used for statistical ( * * 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 of PC with high or low CTH expression . statistical significance was determined using χ2 .

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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.


( A ) Western blot of CTH level in PC3 - T2 with CTH overexpression by pCMV - CTH - HA or PC3 - B2 with CTH knockdown by siCTH - 1 .

(A) Western blot analysis of the CTH level in PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1.


( B ) Top : H2S production capacity of lysates from PC3 - T2 with CTH overexpression by pCMV - CTH - HA or PC3 - B2 with CTH knockdown by siCTH - 1 . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : level of H2S production was quantified by densitometry , histograms represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical ( * P < 0 . 05 ) .

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(B) Top: The H2S production capacity of cell lysates from PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. 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).


( C ) PC3 - B2 were transfected with control or siCTH - 1 then were subjected to migration ( 16h ) invasion ( 24h ) assays . Data information : Data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

(C) PC3-B2 cells were transfected with control or siCTH-1 and then were subjected to migration (16h) and invasion (24h) assays. Data information: 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; ***P<0.001).


( D ) proliferation rates of PC3 - B2 with CTH knockdown . proliferation was performed by MTS reagent . Data information : Data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

(D) Cell proliferation rates of PC3-B2 cells with CTH knockdown. Cell proliferation assay was performed by MTS reagent. Data information: 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; ***P<0.001).


( E ) Western blot of CTH in PC3 transfected with HA - CTH or pCMV - HA vector control .

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(E) Western blot analysis of the CTH levels in PC3 cells transfected with HA-CTH or pCMV-HA vector control.


( F ) PC3 were transfected with HA - CTH or pCMV - HA vector control then incubated for migration ( 16h ) invasion ( 24h ) . Data information : Data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

(F) PC3 cells were transfected with HA-CTH or pCMV-HA vector control and then incubated for migration (16h) and invasion assay (24h). Data information: 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; ***P<0.001).


( G ) PC3 were pre - treated with 10μM PAG for 16 hrs , incubated for migration ( 16h ) invasion ( 24h ) in presence of 10μM PAG . Data information : Data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(G) PC3 cells were pre-treated with 10μM PAG for 16 hrs, and incubated for migration (16h) and invasion assay (24h) in the presence of 10μM PAG. Data information: 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; ***P<0.001).


( A ) Real - Time RT - PCR for IL - 1β mRNA in PC3 with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : data shown represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(A) Real-Time RT-PCR analysis for IL-1β mRNA levels in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown 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; **P<0.01; ***P<0.001).


( B ) ELISA for IL - 1β level in culture medium of PC3 with control or CTH knockdown . Data information data shown represent means ± SD ( n = 3 biological replicates ) . Student ' s t - ( B ) was used for statistical . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(B) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with control or CTH knockdown. Data information All data shown represent the means ± SD (n=3 biological replicates). Student's t-test (B) was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


( C ) PC3 transfected with control or CTH knockdown were incubated for invasion ( 24h ) with recombinant IL - 1β ( 10ng / ml ) added to medium of lower chamber . Data information : data shown represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(C) PC3 cells transfected with control or CTH knockdown were incubated for invasion (24h) assay with recombinant IL-1β (10ng/ml) added to the medium of the lower chamber. Data information: All data shown 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; **P<0.01; ***P<0.001).


( D ) PC3 transfected with control or CTH siRNA were exposed to IL - 1β ( 20ng / ml ) for 1hr . Subcellular localization of p65 was detected by immunocytochemistry . Nuclei were counterstained with DAPI . representative are shown . Scale bars : 25μm .

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(D) PC3 cells transfected with control or CTH siRNA were exposed to IL-1β (20ng/ml) for 1hr. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 25μm.


( E ) of translocation for p65 was scored by counting number of positive stained of p65 to total number of in random microscopic fields . Data information : data shown represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(E) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields. Data information: All data shown 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; **P<0.01; ***P<0.001).


Real - Time RT - PCR for VEGF ( F ) mRNA level in PC3 with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : data shown represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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Real-Time RT-PCR analysis for VEGF (F) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown 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; **P<0.01; ***P<0.001).


Real - Time RT - PCR for MMP - 13 ( G ) mRNA level in PC3 with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : data shown represent means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc was used for statistical . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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Real-Time RT-PCR analysis for MMP-13 (G) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown 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; **P<0.01; ***P<0.001).


( A ) PC3 were incubated for migration ( 16h ) invasion ( 24h ) . DMEM / 10 % FBS , together with various of NaHS ( 10nM - 1mM ) acting as a chemoattractant Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(A) PC3 cells were incubated for migration (16h) and invasion (24h) assay. DMEM/10% FBS, together with various concentration of NaHS (10nM-1mM) acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( B ) PC3 transfected with control or CTH siRNA were incubated for invasion ( 24h ) . DMEM / 10 % FBS , with or without NaHS ( 1μM ) , acting as a chemoattractant Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(B) PC3 cells transfected with control or CTH siRNA were incubated for invasion (24h) assay. DMEM/10% FBS, with or without NaHS (1μM), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( C ) Top : PC3 treated with 100 μM NaHS for 30 min at 37 ° C . were lyzed treated with or without DTT for 10min at 4 ° C before subjecting to modified biotin switch with antibody against p65 to detect S - sulfhydration . Down : quantitative of SSH - p65 level , normalized with total p65 level Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(C) Top: PC3 cells treated with 100 μM NaHS for 30 min at 37°C. Cells were lyzed and treated with or without DTT for 10min at 4°C before subjecting to the modified biotin switch assay with the antibody against p65 to detect S-sulfhydration. Down: quantitative analysis of SSH-p65 protein level, and normalized with total p65 level Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( D ) PC3 were incubate for invasion for 24 hrs . DMEM / 10 % FBS , together with 1μM NaHS an NF - κB inhibitor ( 50μg / ml SN50 , or 100nM QNZ ) , acting as a chemoattractant Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(D) PC3 cells were incubate for invasion assay for 24 hrs. DMEM/10% FBS, together with 1μM NaHS and an NF-κB inhibitor (50μg/ml SN50, or 100nM QNZ), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( E ) Real - Time RT - PCR for IL - 1β , VEGF , MMP - 13 mRNA in PC3 treated with 1μM NaHS for 24h Data information Data shown represent means ± SD ( n = 3 biological replicates ) Student ' s t - tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(E) Real-Time RT-PCR analysis for IL-1β, VEGF, and MMP-13 mRNA levels in PC3 cells treated with 1μM NaHS for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) Student's t-tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( F ) ELISA for IL - 1β level in culture medium of PC3 with various concentrations of NaHS ( 10nM - 10μM ) added for 24h Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(F) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with various concentrations of NaHS (10nM-10μM) added for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


( G ) Western blot of p65 expression in PC3 with p65 knock - out then transfected with p65 wild - type or C38S mutant Data information Data shown represent means ± SD ( n = 3 biological replicates )

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(G) Western blot analysis of the p65 expression in PC3 cells with p65 knock-out and then transfected with p65 wild-type or C38S mutant Data information Data shown represent the means ± SD (n=3 biological replicates)


( H ) PC3 with p65 knock - out with or without overexpression of p65 wild - type or C38S mutant were incubated for invasion for 24 hrs . DMEM / 10 % FBS was used as a chemoattractant Data information Data shown represent means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for statistical ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 )

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(H) PC3 cells with p65 knock-out with or without overexpression of p65 wild-type or C38S mutant were incubated for invasion assay for 24 hrs. DMEM/10% FBS was used as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)


1x106 PC3 with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant inoculated into bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis . ( I ) show H & E of lungs from mice injected with p65 knockout PC3 re - expressing p65 wild - type or C38S mutant . Red arrows indicate metastatic nodules . Scale bar :

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1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis. (I) Images show H&E staining of the lungs from mice injected with p65 knockout PC3 cells re-expressing p65 wild-type or C38S mutant. Red arrows indicate metastatic nodules. Scale bar: 1mm


1x106 PC3 with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant inoculated into bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis total metastatic nodules per lung ( J were quantified . Data are presented as means ± ( n = 7 mice per group ) . Student ' s t - was used for statistical ( * P < 0 . 05 )

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1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis The total metastatic nodules per lung (J were quantified. Data are presented as means ± SEM (n=7 mice per group). Student's t-test was used for the statistical analysis (*P<0.05)


1x106 PC3 with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant inoculated into bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis of metastatic per lung ( K ) were quantified . Data are presented as means ± ( n = 7 mice per group ) . Student ' s t - was used for statistical ( * P < 0 . 05 )

1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis percentage of the metastatic area per lung (K) were quantified. Data are presented as means ± SEM (n=7 mice per group). Student's t-test was used for the statistical analysis (*P<0.05)


( A ) Western blot of CTH expression in PC3 stably expressing control or CTH shRNA .

(A) Western blot analysis of the CTH expression in PC3 cells stably expressing control or CTH shRNA.