Representative of colony forming capacity at 14 days determined using crystal violet in empty vector ( EV ) CEP55 - overexpressing ( CEP55 # 17 CEP55 # 18 ) lines .

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Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector (EV) and CEP55-overexpressing (CEP55#17 and CEP55#18) lines.


Representative of single plane phase - contrast Z - stacked immunofluorescence of empty vector ( EV ) CEP55 - overexpressing MCF10A grown on Matrigel for 14 days . Red : Cytokeratin - 19 ; Green : F - actin stained by Phallodin Blue : DAPI . Z - stack were acquired through Zeiss LSM 780 confocal microscope - ZMBH .

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Representative images of single plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: Cytokeratin-19; Green: F-actin stained by Phallodin and Blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH.


( A ) Polyploidy ( > 4N contents ) determined using FACS in control CEP55 knockdown MDA - MB - 231 ( left panel ) . Representative corresponding cytogram showing different phases of cycle polyploidy subpopulation analyzed using ModFit LT 4 . 0 software ( right panel ) . Yellow peaks represent subpopulation of polyploidy / aneuploidy . Graph represents mean ± of three independent experiments .

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(A) Polyploidy analysis (>4N DNA contents) determined using FACS in control and CEP55 knockdown MDA-MB-231 cells (left panel). Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4.0 software (right panel). Yellow peaks represent subpopulation of polyploidy/aneuploidy. Graph represents the mean±SEM of three independent experiments.


( B ) of chromosome number in control CEP55 knockdown MDA - MB - 231 ( sh # 8 ) by Giemsa of metaphase spreads ( left panel ) . Representative showing chromosome spreads assessed by Zeiss AxioScope2 microscopy ( right panel ) . n = number of metaphase spreads counted ( mean ± 95 % confidence interval of two independent experiments ) .

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(B) Analysis of chromosome number in control and CEP55 knockdown MDA-MB-231 cells (sh#8) by Giemsa staining of metaphase spreads (left panel). Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy (right panel). n= number of metaphase spreads counted (mean ±95% confidence interval of two independent experiments).


( C ) Representative SNP array plots showing chromosomal alterations following CEP55 - knockdown in MDA - MB - 231 . BAF is B allele frequency , value between 0 1 represents proportion contributed by one SNP allele ( B ) to total copy number : BAF is an estimate of NB / ( NA + NB ) , where NA NB are number of A B alleles , respectively .

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

(C) Representative SNP array plots showing chromosomal alterations following CEP55-knockdown in MDA-MB-231 cells. BAF is B allele frequency, value between 0 and 1 and represents the proportion contributed by one SNP allele (B) to the total copy number: BAF is an estimate of NB/(NA+ NB), where NA and NB are the number of A and B alleles, respectively.


( D ) of genome altered evaluated using amplified copies ( 6 - 8 copies across genome ) in both parental CEP55 knockdown determined using data from SNP arrays . Graph represents mean ± of two independent experiments .

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(D) Percentage of genome altered evaluated using amplified copies (6-8 copies across genome) in both parental and CEP55 knockdown cells determined using data from SNP arrays. Graph represents the mean±SEM of two independent experiments.


( E ) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A . Red box indicates an extra chromosome 20 in CEP55 - overexpressing .

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(E) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells. Red box indicates an extra chromosome 20 in CEP55-overexpressing cells.


( G ) Kaplan - Meier survival of METABRIC data showing relationship between CEP55 expression survival . Cases were subdivided into tertiles . Log - rank ( Mantel - Cox ) , P < 0 . 0001 .

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(G) Kaplan-Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival. Cases were subdivided into tertiles. Log-rank (Mantel-Cox) test, P< 0.0001.


( A ) Representative cytogram of control CEP55 knockdown MDA - MB - 231 showing cycle profiles following treatment with without B12536 ( 5 nM ) .

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(A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM).


of polyploidy ( > 4N ) following B12536 ( 5 nM ) in control CEP55 knockdown MDA - MB - 231 . Graph represents mean ± of three independent experiments .

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Percentage of polyploidy (>4N) following B12536 (5 nM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.


of polyploidy ( > 4N ) following nocodazole ( 0 . 5 µM ) in control CEP55 knockdown MDA - MB - 231 . Graph represents mean ± of three independent experiments .

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Percentage of polyploidy (>4N) following nocodazole (0.5 µM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.


Apoptotic fraction ( Sub - G1 population ) determined by propidium iodide of treated with PLK1i Graph represents mean ± of three independent experiments .

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Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with PLK1i Graph represents the mean±SEM of three independent experiments.


Apoptotic fraction ( Sub - G1 population ) determined by propidium iodide of treated with Nocodazole Graph represents mean ± of three independent experiments .

Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean±SEM of three independent experiments.


( F ) of sub - G1 fraction following B12536 ( 10 nM ) or nocodazole ( 0 . 5 µM ) in control CEP55 knockdown Hs578T . Graph represents mean ± of two independent experiments .

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(F) Percentage of sub-G1 fraction following B12536 (10 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown Hs578T cells. Graph represents the mean±SEM of two independent experiments.


( G ) Representative cytogram of empty vector ( EV ) CEP55 - overexpressing MCF10A showing cycle profile following treatment with without B12536 ( 10 nM ) .

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(G) Representative cytogram of empty vector (EV) and CEP55-overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 (10 nM).


( H ) of polyploidy following treatment with without B12536 ( 10 nM ) in empty vector ( EV ) CEP55 - overepressing MCF10A . Graph represents mean ± of three independent experiments .

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(H) Percentage of polyploidy following treatment with and without B12536 (10 nM) in empty vector (EV) and CEP55-overepressing MCF10A cells. Graph represents the mean±SEM of three independent experiments.


( I ) Corresponding sub - G1 population determined using propidium iodide of treated with B12536 as described in panel H . Graph represents mean ± of three independent experiments .

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(I) Corresponding sub-G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H. Graph represents the mean±SEM of three independent experiments.


( A ) Box whiskers plot showing average time spent in mitosis ( B ) mitotic outcomes in control CEP55 knockdown MDA - MB - 231 following treatment with BI2536 ( 5 nM ) . Time taken to complete mitosis was defined as time from envelope breakdown until two daughter were observed whereas mitotic slippage or was defined as that prematurely exited mitosis with a flattened a multinucleated phenotype or died during mitosis , characterized by blebbing . Graph represents mean ± of two independent experiments . For each experiments n = 50 mitotic were counted per condition using Olympus Xcellence IX81 time - lapse microscopy .

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

(A) Box and whiskers plot showing average time spent in mitosis and (B) mitotic outcomes in control and CEP55 knockdown MDA-MB-231 cells following treatment with the BI2536 (5 nM). Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis, characterized by membrane blebbing. Graph represents the mean±SEM of two independent experiments. For each experiments n=50 mitotic cells were counted per condition using Olympus Xcellence IX81 time-lapse microscopy.


( C , D ) were pre - treated for two hours with 50 µM of pan Caspase inhibitor Z - VAD - FMK , followed by treatment with B12536 ( 5 nM ) , before assessment of fate ( C ) an average time spent in mitosis ( D ) Graph represents mean ± of two independent experiments .

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(C, D) Cells were pre-treated for two hours with 50 µM of the pan Caspase inhibitor Z-VAD-FMK, followed by treatment with B12536 (5 nM), before assessment of cell fate (C) and an average time spent in mitosis (D) Graph represents the mean±SEM of two independent experiments.


( E ) were synchronized using double thymidine then released into nocodazole ( 0 . 5 µM ) , lysates were collected at indicated time points . Immunoblot was then performed to determine expression activity of mitotic regulators as indicated . of phospho - MEKT286 dephosphorylation of phospho - CDK1Y15 served as markers of Cdk1 activation / mitotic entry . COX - IV served as a loading control .

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(E) Cells were synchronized using double thymidine then released into nocodazole (0.5 µM), and protein lysates were collected at the indicated time points. Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of Cdk1 activation/mitotic entry. COX-IV served as a loading control.


( G ) Representative mCerulean lifetimes maps of Hs578T control CEP55 knockdown upon nocodazole ( 0 . 5 µM ) treatment at indicated time points . were synchronized using double thymidine for 16 hours prior to nocodazole treatment .

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(G) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole (0.5 µM) treatment at indicated time points. Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment.


( H ) of mCerulean lifetimes of control Hs578T CEP55 knockdow ncells in response to treatment with nocodazole at indicated time points as shown in G . Blue : 1 ns ; red : 3 ns . Graph represents mean ± of three independent experiments .

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(H) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G. Blue: 1 ns; red: 3 ns. Graph represents the mean±SEM of three independent experiments.


( I ) Control CEP55 knockdown MDA - MB - 231 were synchronized as above , released into culture medium for six hours then blocked with nocodazole ( 0 . 5 µM ) for an additional four hours prior to treatment with Cdk1 inhibitor RO - 3306 for an additional 16 hours . Sub - G1 were then identified by propidium iodide quantified by FACS . Graph represents mean ± of two independent experiments .

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(I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments.


( J ) Control CEP55 knockdown MDA - MB - 231 were transfected with MAD2 siRNA ( 5 nM ) for 48 hours followed by nocodazole ( 0 . 5 µM ) treatment for an additional 12 hours . were collected , fixed sub - G1 was performed as described in methods . Graph represents mean ± of two independent experiments .

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

(J) Control and CEP55 knockdown MDA-MB-231 cells were transfected with MAD2 siRNA (5 nM) for 48 hours followed by nocodazole (0.5 µM) treatment for an additional 12 hours. Cells were collected, fixed and sub-G1 analysis was performed as described in methods. Graph represents the mean±SEM of two independent experiments.


( A ) Immunoblots was performed to determine CEP55 in MDA - MB - 231 treated with multiple inhibitors targeting EGFR / HER2 pathway for 24 hour . following inhibitors were used : MEK1 / 2i ( AZD6244 ( 1 µM ) ) , AKT , PI3K / mTORi ( BEZ235 ( 0 . 5 µM ) , AKTi VIII ( 1 µM ) ) , EGFRi HER2i ( Erlotinib ( 1 µM ) , Afatinib ( 0 . 25 µM ) , Lapatinib ( 1 µM ) , Trastuzumab ( 10 µg / ml ) or pan VEGFR , PDGFR RAF kinases ( Sorafinib , ( 1 µM ) ) . COX - IV as a loading control .

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(A) Immunoblots analysis was performed to determine CEP55 levels in MDA-MB-231 cells treated with multiple inhibitors targeting the EGFR/HER2 pathway for 24 hour. The following inhibitors were used: MEK1/2i (AZD6244 (1 µM)), the AKT, PI3K/mTORi (BEZ235 (0.5 µM), AKTi VIII (1 µM)), EGFRi and HER2i (Erlotinib (1 µM), Afatinib (0.25 µM), Lapatinib (1 µM), Trastuzumab (10 µg/ml) or the pan VEGFR, PDGFR and RAF kinases (Sorafinib, (1 µM)). COX-IV as a loading control.


( B ) Immunoblots of MDA - MB - 231 treated with three different MEK1 / 2 inhibitors at various concentrations ( Selumetinib AZD6244 , Trametinib GSK1120212 Binimetinib MEK162 ) after 24h . COX - IV as a loading control .

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(B) Immunoblots analysis of MDA-MB-231 cells treated with three different MEK1/2 inhibitors at various concentrations (Selumetinib AZD6244, Trametinib GSK1120212 and Binimetinib MEK162) after 24h. COX-IV as a loading control.


( C ) Relative CEP55 promoter luciferase activity in MDA - MB - 231 either treated with AZD6244 ( 1 µM ) for 6 hours or ERK1 / 2 knockdown for 24 hours . PGL basic reporter plasmid was used to normalize basal CEP55 promoter activity . Graph represents mean ± of two independent experiments .

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(C) Relative CEP55 promoter luciferase activity in MDA-MB-231 cells either treated with AZD6244 (1 µM) for 6 hours or ERK1/2 knockdown for 24 hours. PGL basic reporter plasmid was used to normalize basal CEP55 promoter activity. Graph represents the mean±SEM of two independent experiments.


( D ) Immunoblot of MDA - MB - 231 cultured in 0 . 1 % bovine serum for 24h stimulated with EGF ( 100 ng / mL ) for indicated time points . Phosphorylation of EGFR , ERK1 / 2 along with CEP55 MYC were determined . COX - IV as a loading control .

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(D) Immunoblot analysis of MDA-MB-231 cells cultured in 0.1% fetal bovine serum for 24h and stimulated with EGF (100 ng/mL) for the indicated time points. Phosphorylation of EGFR, ERK1/2 along with CEP55 and MYC were determined. COX-IV as a loading control.


( E ) Relative CEP55 promoter luciferase activity upon MYC siRNA was determined using DualGlo in MDA - MB - 231 similar to experiment in panel C . Graph represents mean ± of two independent experiments .

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(E) Relative CEP55 promoter luciferase activity upon MYC siRNA was determined using DualGlo assay in MDA-MB-231 cells similar to experiment in panel C. Graph represents the mean±SEM of two independent experiments.


( F ) Immunoblots showing MYC CEP55 expression in MDA - MB - 231 upon transfection with siRNA against MYC , ETS1 ( 10 nM ) , with without EGF ( 100 ng / mL ) stimulation as indicated time points . COX - IV served as a loading control .

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(F) Immunoblots analysis showing MYC and CEP55 expression in MDA-MB-231 cells upon transfection with siRNA against MYC, and ETS1 (10 nM), with and without EGF (100 ng/mL) stimulation as indicated time points. COX-IV served as a loading control.


( G ) Immunoblots showing CEP55 MYC following 4 - hydroxytamoxifen ( 4OHT ) ( 0 . 5 µM ) induction in MCF10A MYCER cultured in 0 . 1 % bovine serum contained media at indicated time points . COX - IV served as a loading control .

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

(G) Immunoblots analysis showing CEP55 and MYC levels following 4-hydroxytamoxifen (4OHT) (0.5 µM) induction in MCF10A MYCER cells cultured in 0.1% fetal bovine serum contained media at indicated time points. COX-IV served as a loading control.


( H ) of Pearson correlation coefficients ( PCC ) of CEP55 vs . MYC with R = 0 . 9982 , p = 0 . 0016 . band intensities as shown in H were measured using ImageJ software .

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(H) Analysis of Pearson correlation coefficients (PCC) of CEP55 vs. MYC levels with R=0.9982, p=0.0016. Protein band intensities as shown in H were measured using ImageJ software.


( I ) of Pearson correlation coefficients ( PCC ) of CEP55 vs . MYC transcriptional activity in breast cancer TCGA data with R = 0 . 4157 , p < 0 . 0001 .

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(I) Analysis of Pearson correlation coefficients (PCC) of CEP55 vs. MYC transcriptional activity in breast cancer TCGA tumor data with R=0.4157, p<0.0001.


( J ) Both control CEP55 knockdown MDA - MB - 231 were exposed with different concentrations of PLK1 ( BI2536 ) alone ( i ) or in combination with AZD6244 ( 1 µM ) ( ii - iv ) , viability was determined after 6 days . dose - response curve was generated by calculating viability relative to untreated control plotted against drug . Graph represents mean ± of three independent experiments .

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(J) Both control and CEP55 knockdown MDA-MB-231 cells were exposed with different concentrations of PLK1 (BI2536) alone (i) or in combination with AZD6244 (1 µM) (ii-iv), and cell viability was determined after 6 days. The dose-response curve was generated by calculating cell viability relative to untreated control and plotted against drug concentration. Graph represents the mean±SEM of three independent experiments.


( K ) of sub - G1 population identified using propidium iodide quantified by FACS following single combination treatment with AZD6244 ( 1 µM ) BI2536 ( 2 . 5 nM ) inhibitors after 96 h . Graph represents mean ± of two independent experiments .

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(K) Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors after 96 h. Graph represents the mean±SEM of two independent experiments.


( L ) Immunoblots of both control CEP55 knockdown MDA - MB - 231 treated with single combination treatments after 96h PARP , Caspase - 3 along with MYC , ERK1 / 2 CEP55 were determined . COX - IV as a loading control .

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(L) Immunoblots analysis of both control and CEP55 knockdown MDA-MB-231 cells treated with single and combination treatments after 96h and cleaved PARP, Caspase-3 along with MYC, ERK1/2 and CEP55 were determined. COX-IV as a loading control.


( A ) Heat map showing relative viability of a panel of human breast cancer lines treated single or in combination with AZD6244 ( 1 µM ) BI2536 ( 2 . 5 nM ) inhibitors viability was determined after 6 days . DMSO treated control was used to calculate of affected by or combination treatment . NN : near normal . Heat map represents data derived from three independent experiments .

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(A) Heat map showing relative cell viability of a panel of human breast cancer lines treated single or in combination with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors and cell viability was determined after 6 days. DMSO treated control was used to calculate percentage of cells affected by individual or combination treatment. NN: near normal. Heat map represents data derived from three independent experiments.


( B ) Immunoblot was performed on MDA - MB - 157 SUM159PT treated with single or in combination with AZD6244 BI2536 inhibitors after 96 hours PARP Caspase - 3 were determined along with CEP55 , MYC both phosphorylated total ERK1 / 2 . COX - IV as a loading control ( left panels ) . of sub - G1 population identified using propidium iodide quantified by FACS following single combination treatment with AZD6244 BI2536 inhibitors after 96h . Graph represents mean ± of two independent experiments ( middle panels ) . Representative of colony forming capacity at 14 days determined using crystal violet in treated with single combination inhibitors ( middle panels ) . Combination index ( CI ) values calculated through CompuSyn software ( right panels ) where 1 indicative of additive ( no interaction ) , > 1 indicative of antagonistic , < 1 indicative of synergistic .

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

(B) Immunoblot analysis was performed on MDA-MB-157 and SUM159PT cells treated with single or in combination with AZD6244 and BI2536 inhibitors after 96 hours and cleaved PARP and Caspase-3 were determined along with CEP55, MYC and both phosphorylated and total ERK1/2. COX-IV as a loading control (left panels). Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 and BI2536 inhibitors after 96h. Graph represents the mean±SEM of two independent experiments (middle panels). Representative images of colony forming capacity at 14 days determined using crystal violet staining in cells treated with single and combination inhibitors (middle panels). Combination index (CI) values calculated through CompuSyn software (right panels) where 1 indicative of additive (no interaction), >1 indicative of antagonistic, and <1 indicative of synergistic.


( C ) of mitotic outcomes determined using time - lapse microscopy of MDA - MB - 231 treated with single or in combination drugs of BI2536 ( 2 . 5 nM ) AZD6244 ( 1 µM ) . Graph represents mean ± of two independent experiments . For each experiments n = 50 were counted per condition .

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(C) Percentage of mitotic outcomes determined using time-lapse microscopy of MDA-MB-231 cells treated with single or in combination drugs of BI2536 (2.5 nM) and AZD6244 (1 µM). Graph represents the mean±SEM of two independent experiments. For each experiments n=50 cells were counted per condition.


( D ) Immunoblot was performed to determine expression activity of mitotic apoptotic regulators upon single combination treatment with AZD6244 BI2536 inhibitors as indicated time points in both MDA - MB - 231 SUM159PT . of phospho - MEKT286 dephosphorylation of phospho - CDK1Y15 served as markers of CDK1 activation / mitotic entry . COX - IV as a loading control .

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(D) Immunoblot analysis was performed to determine the expression and activity of mitotic and apoptotic regulators upon single and combination treatment with AZD6244 and BI2536 inhibitors as indicated time points in both MDA-MB-231 and SUM159PT cells. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of CDK1 activation/mitotic entry. COX-IV as a loading control.


( A ) Left , Six week - old BALB / c cohorts of mice were injected in 4th inguinal mammary fat pad with Cep55 - ovexpressing mammary carcinoma 4T1 . 2 . ( , mm2 ) was measured using a digital calliper mean of each cohort is presented . Mice were treated with vehicle , AZD6244 ( 12 . 5mg / kg BID ) , BI6727 ( 12 . 5mg / kg thrice weekly ) , or combined AZD6244 BI2536 treatment . Right , representative excised are shown . Graph represents mean ± from six mice / group .

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(A) Left, Six week-old female BALB/c cohorts of mice were injected in the 4th inguinal mammary fat pad with the Cep55-ovexpressing mammary carcinoma cell line 4T1.2. Tumor size (area, mm2) was measured using a digital calliper and mean tumor size of each cohort is presented. Mice were treated with vehicle, AZD6244 (12.5mg/kg BID), BI6727 (12.5mg/kg thrice weekly), or combined AZD6244 and BI2536 treatment. Right, representative excised tumor images are shown. Graph represents the mean tumor area±SEM from six mice/group.


( B ) Immunoblot was performed on lysates to determine target inhibition following single combined treatments as indicated in panel A . Caspase - 3 was probed to determine apoptosis . GAPDH as a loading control .

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(B) Immunoblot analysis was performed on tumor lysates to determine target inhibition following single and combined treatments as indicated in panel A. Cleaved Caspase-3 was probed to determine apoptosis. GAPDH as a loading control.


( C ) Six week - old BALB / c cohorts of mice were pre - treated with single combination BI6727 AZD6244 inhibitors as indicated in panel A for 4 days , followed by 4T1 . 2 were injected in 4th inguinal mammary fat pad . Survival of mice was then monitored over indicated period of time significant of data was analyzed by log - rank ( P = 0 . 0005 ) ; n = 6 mice / group .

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(C) Six week-old female BALB/c cohorts of mice were pre-treated with single and combination BI6727 and AZD6244 inhibitors as indicated in panel A for 4 days, followed by 4T1.2 cells were injected in the 4th inguinal mammary fat pad. Survival of the mice was then monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P=0.0005); n=6 mice/group.


( D ) Growth rate ( mean , , mm2 ) of MDA - MB - 231 xenografts in BALB / c nude mice treated with vehicle , AZD6244 , BI6727 , or combined AZD6244 BI6727

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(D) Growth rate (mean tumor size, area, mm2) of MDA-MB-231 xenografts in BALB/c nude mice treated with vehicle, AZD6244, BI6727, or combined AZD6244 and BI6727 treatment


( E ) survival of mice was monitored over indicated period of time significant of data was analyzed by log - rank ( P < 0 . 0001 ) ; n = 6 mice / group .

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(E) survival of mice was monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P<0.0001); n=6 mice/group.


In vitro pelleting comparing TRAPPIII versus subunits . While both complexes exhibit a small amount of pelleting in absence of synthetic liposomes , in their presence only TRAPPIII is enriched in - bound fraction . ( S = supernatant , P = pellet ) Representative of n = 3 independent experiments . of D . Average % binding between three assays is plotted , error bars represent 95 % confidence intervals . value for each replicate is depicted . negative values arise from subtraction of average amount of that pellets in absence of liposomes . Analyzed by unpaired , two - Student ' s t - with Welch ' s correction , * p = 0 . 0229 . n = 3 independent experiments .

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In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits. While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes, in their presence only the TRAPPIII complex is enriched in the membrane-bound fraction. (S=supernatant, P=pellet) Representative of n=3 independent experiments.   Quantification of D. Average % membrane binding between three assays is plotted, the error bars represent the 95% confidence intervals. The value for each replicate is depicted. The negative values arise from subtraction of the average amount of protein that pellets in the absence   of liposomes. Analyzed by unpaired, two-tailed Student's t-test with Welch's correction, * p = 0.0229. n=3 independent experiments.


Representative Coomassie - stained gel of exclusion chromatography fractions after purification of recombinant wild - type TRAPPIII . Representative of n = 3 independent experiments . As in A , using a Trs85 truncation containing only final 198 . NOTE : Trs85 [ 501 - 698 ] is ~ 25kD in now migrates at same as some of subunits . species migrating near 85kD are contaminants present due to lower expression level of this mutant construct . Representative of n = 3 independent experiments . As in A / B , using a Trs85 mutant with a truncation of loop from 575 - 603 . Trs85 [ ∆ 575 - 603 ] appears to dissociate from during chromatography , indicating a reduced affinity for subunits . Representative of n = 3 independent experiments .

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Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.   As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.   As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.


Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with same Trs85 mutants . Representative of n = 3 independent experiments . ( WCE = whole extract , WT = wild - type , EV = empty vector )

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Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)


Live microscopy of Trs85 - mNeonGreen mRFPmars - Ypt1 . Localization of Rab1 / Ypt1 to punctate structures is indicative of its activation by TRAPPIII . Scale bar is 2 microns . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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Live cell fluorescence microscopy of Trs85-mNeonGreen and mRFPmars-Ypt1. Localization of Rab1/Ypt1 to punctate structures is indicative of its activation by TRAPPIII. Scale bar is 2 microns. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


E . of colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents MOC for a cropped image containing 2 - 9 . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons , * * * * p < 0 . 0001 . F . of colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . of wild - type Rab1 / Ypt1 HVD is in black , substituted residues are colored to correspond with their location on graph . " CIM " motif is required for enzymatic prenylation of Rab , but these chimeras are anchored to plasma independently of prenylation , via Fis1 - fusion . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents MOC for a cropped image containing 2 - 9 . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons , but significance indicators are only present for each substitution mutant compared to " wild - type " Ypt31HVDYpt1 : vs . 181TTQ p = 0 . 9924 , vs . 184KKE p = 0 . 9997 , vs . 187DKGN * p = 0 . 0145 , vs . 191VNL , 194KGQS , 198LTNT * * * * p < 0 . 0001 , ns = not significant .

O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, **** p < 0.0001. F. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. The sequence of wild-type Rab1/Ypt1 HVD is in black, the substituted residues are colored to correspond with their location on the graph. The "CIM" motif is required for enzymatic prenylation of the Rab, but these chimeras are anchored to the plasma membrane independently of prenylation, via Fis1-protein fusion. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, but significance indicators are only present for each substitution mutant compared to "wild-type" Ypt31HVDYpt1: vs. 181TTQ p = 0.9924, vs. 184KKE p = 0.9997, vs. 187DKGN * p = 0.0145, vs. 191VNL, 194KGQS, 198LTNT **** p < 0.0001, ns = not significant.


Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with same Trs85 substitution mutants . Representative of n = 3 independent experiments . ( WCE = whole extract , WT = wild - type , EV = empty vector )

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Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 substitution mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)


Live microscopy of Trs85 - mNeonGreen substitution mutants mRFPmars - Ypt1 . Scale bar = 2 μm . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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Live cell fluorescence microscopy of Trs85-mNeonGreen substitution mutants and mRFPmars-Ypt1. Scale bar = 2 μm. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


GFP - Atg8 processing is monitored for either TRAPP binding mutations or amphipathic helix ( binding ) substitutions . With functional Trs85 , there is an accumulation of free GFP . Samples are from pre - rapamycin treatment ( - ) 1hr after addition of rapamycin ( + ) . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector ) of A . average of at least three replicates is plotted with error bars representing 95 % confidence interval . of free GFP for wild - type Trs85 is compared to each mutant using an unpaired , two - Student ' s t - with Welch ' s correction : vs . EV * * p = 0 . 0022 , vs . R618E , R619E , R620E , K621E * * * p = 0 . 0008 , vs . R618A , R619A , R620A , K621A * * * p = 0 . 0006 , vs . 501 - 698 * * * p = 0 . 0003 , vs . 4A p = 0 . 3295 , vs . 7A * * * p = 0 . 0008 , vs . 4E * * * p = 0 . 0005 , ns = not significant . ( WT = wild - type , EV = empty vector )

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GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)   Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p =   0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector)


a , COS7 co - transfected with GFP - ATG14 DsRed - ER or RFP - SEC61b ; ( an ER marker ) were immunostained for 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 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 stably expressing GFP - DFCP1 . Cyto , cytosol ; micro , microsome ; mito , mitochondria ; PNS , post - 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 stably expressing yellow ( YFP ) - ATG5 were transfected with RFP - SEC61b ; teal ( TFP ) - mito , 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 of COS7 transfected with YFP - ATG5 , RFP - SEC61b ; 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 surface of ER ( c ) to surface of mitochondria ( d ) . e , f , Distances of YFP - ATG5 dots over their lifetime for data from c ( e ) d ( f ) , respectively . 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 expressing GFP - SEC61b ; STX17 were immunostained for TOMM20 .

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


b , HeLa 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 .

c, Immunoblot of subcellular fractions of HEK293 cells.


d , STX17 knockdown COS7 expressing GFP - ATG14 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 STX17 - 2 ) HEK293 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 ATG14 using HeLa expressing Flag - ATG14 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 from HeLa expressing Myc - STX17 or Myc . Scale bars , 5 µm ( a , b ) 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 electron microscopy of STX17 knockdown HeLa expressing GFP - LC3 ( arrows ) infected with ( 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 of STX17 knockdown . arrows denote autolysosomes , black arrows denote autophagosome , 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 of STX17 knockdown HeLa 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 . e , of 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 . 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 in STX17 knockdown . St , starved ; wor , wortmannin . error bars denote s . d . Scale bars , 1 µm ( a , b ) , 200 nm ( c ) 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 .

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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 is co - localized with γH2AX . Immunofluorescence studies using U2OS19 ptight13 GFP - LacR containing a stably integrated I - SceI cleavage site flanked by 256 copies of lac operator ( lacO ) on one side 96 copies of tetracycline response element on other side ( tetO ) . localization of GFP - lac repressor ( GFP - LacR ) at lac - operator sequences in nucleus before ( − I - SceI ) 16 h after I - SceI - induced ( + I - SceI ) DSB . After 16 h of doxy treatment , CHD1 γH2AX co - localized at I - SceI cleavage site , along with - bound GFP - LacR but not in uninduced ( − 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 ) of co - localization of CHD1 with lac array , mean values of three independent experiments counting at least 100 are represented in 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 were treated with NCS ( 100 ng / ml ) for 2 h with EdU for 45 min . were stained with EdU first then proximity ligation ( PLA ) was performed using γH2AX 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 ) of PLA signal from ( D ) in EdU - positive - negative using ImageJ . For more than 100 were analyzed for each condition 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 upon double strand break induction . PC3 with stable control ( shCont ) or CHD1 shRNA ( shCHD1 ) expression were treated with NCS for indicated time points fractions were immunoblotted with CHD1 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 with stable expression of either control ( shCont ) or CHD1 shRNA ( shCHD1 ) were treated with γ - radiation ( 3 Gy ) after 1 h 24 h were immunostained for γH2AX ( A ) number of γH2AX foci per were determined for each time point , more than 50 were counted in each condition ( B ) . Scale bar 5 µm . mean values of three independent experiments ( ± SD ) are shown . p - values ( 0 . 0008 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 show prolonged γH2AX accumulation . PC3 with shCont or shCHD1 were treated with γ - radiation as in ( A ) total 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 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 .

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

(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 .

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

(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 .

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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 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 .

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

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 .

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

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 .

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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 ) 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 .

B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-DISEASE B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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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 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 ) .

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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 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 B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-DISEASE O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-DISEASE O O O O O B-DISEASE O O O O O O O O B-SMALL_MOLECULE O O O B-DISEASE O O O O O B-DISEASE O O O O O O O O B-SMALL_MOLECULE O O O B-DISEASE O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O B-DISEASE B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE B-TISSUE O O O O O O O B-TISSUE O O O O O O B-DISEASE O O O O O O O O O O O B-DISEASE O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 ) .

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

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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 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).