Figure 5 Figure 5 . EGF treatment dissociates DENND2B from ITSN A HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for the indicated times and cell lysates were analyzed by Western blot . B Quantification of A where P - PKD is normalized to Hsc70 . Mean + / - SD . ANOVA with Dunnett post - test * p = 0 . 015 and * * p = 0 . 003 n = 3 from 3 independent experiments . C HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti - Flag . Bound proteins were detected by Western blot . D Quantification of B where P - PKD substrate is normalized to Flag - DENND2B . Mean + / - SD Welch ' s t - test * p = 0 . 036 , n = 4 from 3 independent experiments . E HEK - 293T cells were serum starved , treated with 100 ng / mL EGF for 5 min and cell lysates were incubated with GST - SH3A . Total proteins and bound proteins were detected by Ponceau S staining and Western blot , respectively . F Quantification of E where bound Flag - DENND2B is normalized to SM . Mean + / - SD , Welch ' s t - test * p = 0 . 012 , n = 6 from 3 independent experiments .

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Figure 5 Figure 5. EGF treatment dissociates DENND2B from ITSN A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot. B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments.C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments.E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments.


Figure 1Wild - type mice have small defined crypts in the small intestine with little nuclearβ - catenin at the bottom of the crypt . The small intestine of AhCreERAPCfl / fl , AhCreERCatnblox ( ex3 ) / lox ( ex3 ) ( day 5 ) or AhCreERGsk3alphafl / fl betafl / fl ( day 6 ) show the crypt - progenitor cell ( CPC ) phenotype with increased crypt size ( red bar ) and nuclearβ - catenin ( arrows ) along the crypt - villus axis . Scale bar , 100 μm . A heterozygous activation of β - catenin ( AhCreER Catnblox ( ex3 ) / + ) shows no increase in crypt size or nuclear β - catenin at days 5 - 10 . At day 15 post - induction , accumulation of nuclear β - catenin ( arrows ) becomes evident with a dramatic CPC phenotype at about day 25 . Scale bar , 100 μm . Culture of small intestinal crypts of WT and VilCreER Apcfl / fl ( or AhCreER Apcfl / fl ) mice with / without R - spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc - deficient spheres . Representative photos were taken at day 5 in culture . Black scale bar , 50 μm . At day 5 post - induction , only crypts from AhCreERCatnblox ( ex3 ) / lox ( ex3 ) but not AhCreERCatnblox ( ex3 ) / + survive in culture without the addition of R - spo1 . At day 10 post - induction , we observed a mixed phenotype of more organoid - like structures in AhCreERCatnblox ( ex3 ) / + compared to spheres from Catnblox ( ex3 ) / lox ( ex3 ) crypts in the first week of culture . Black scale bar , 50 μm . Quantification of organoids / spheres at day 10 post - induction ( N = 2 or 3 mice per genotype , mean of 6 technical replicates per mouse ) .

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Figure 1Wild-type mice have small defined crypts in the small intestine with little nuclearβ-catenin at the bottom of the crypt. The small intestine of AhCreERAPCfl/fl, AhCreERCatnblox(ex3)/lox(ex3) (day 5) or AhCreERGsk3alphafl/fl betafl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclearβ-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm.A heterozygous activation of β-catenin (AhCreER Catnblox(ex3)/+) shows no increase in crypt size or nuclear β-catenin at days 5-10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm.Culture of small intestinal crypts of WT and VilCreER Apcfl/fl (or AhCreER Apcfl/fl) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc-deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm.At day 5 post-induction, only crypts from AhCreERCatnblox(ex3)/lox(ex3) but not AhCreERCatnblox(ex3)/+ survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCreERCatnblox(ex3)/+ compared to spheres from Catnblox(ex3)/lox(ex3)crypts in the first week of culture. Black scale bar, 50 μm.Quantification of organoids/spheres at day 10 post-induction (N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).


Figure 2Table shows that only homozygous activation of β - catenin ( Lgr5CreER Catnblox ( ex3 ) / lox ( ex3 ) ) or loss of Apc ( Lgr5CreER Apcfl / fl ) in Lgr5 - positive stem cells results in colonic lesions in cohorts , sampled when signs of sickness were apparent . There were no lesions observed when only one allele of β - catenin ( Lgr5CreER Catnblox ( ex3 ) / + ) was mutated ( chi - squared test , P < 0 . 001 ) . Colonic lesions were scored from H & amp ; amp ; E by identification of disorganised epithelium . Staining for β - catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions . Scale bar , 100 μm .

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Figure 2Table shows that only homozygous activation of β-catenin (Lgr5CreER Catnblox(ex3)/lox(ex3)) or loss of Apc (Lgr5CreER Apcfl/fl) in Lgr5-positive stem cells results in colonic lesions in cohorts, sampled when signs of sickness were apparent. There were no lesions observed when only one allele of β-catenin (Lgr5CreER Catnblox(ex3)/+) was mutated (chi-squared test, P < 0.001).Colonic lesions were scored from H&amp;E by identification of disorganised epithelium. Staining for β-catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions. Scale bar, 100 μm.


Figure 3Staining of small intestinal and colonic crypts for E - cadherin . Scale bar , 100 μm . Western blot of purified crypts from the small intestine and colon ( N = 4 mice ) . qRT - PCR of whole tissue from small intestine and colon . Expression of mRNA ( 2 ( − dCt ) ) calculated relative to GAPDH ( N = 3 mice ) . Statistics : one - sided Mann - Whitney U - test . Proximity ligation assay for E - cadherin and β - catenin . For each mouse ( N = 6 ) , at least 10 crypts per tissue were analysed to calculate the mean . Statistics : one - sided Mann - Whitney U - test ; white scale bar , 50 μm .

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Figure 3Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm.Western blot of purified crypts from the small intestine and colon (N = 4 mice).qRT-PCR of whole tissue from small intestine and colon. Expression of mRNA (2(−dCt)) calculated relative to GAPDH (N = 3 mice). Statistics: one-sided Mann-Whitney U-test.Proximity ligation assay for E-cadherin and β-catenin. For each mouse (N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann-Whitney U-test; white scale bar, 50 μm.


Figure 4Immunoprecipitation ( IP ) of E - cadherin from VilCreER Catnblox ( ex3 ) / lox ( ex3 ) mice , 24 h ( day 1 , top ) and 48 h ( day 2 , bottom ) after induction . The ratio of wild - type ( WT , top lane ) and mutant exon 3 β - catenin ( Δex3 , bottom lane ) bound to E - cadherin ( IP : Ecad ) was calculated for each tissue . Graphs show average of experiments ( N = 3 mice for each genotype and time point ) ; error bars represent s . e . m . IP of E - cadherin from VilCreER Catnblox ( ex3 ) / + mice at day 4 and day 8 . Graphs show average of experiments ( N = 3 mice for each genotype and time point ) ; error bars represent s . e . m .

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Figure 4Immunoprecipitation (IP) of E-cadherin from VilCreER Catnblox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.IP of E-cadherin from VilCreER Catnblox(ex3)/+ mice at day 4 and day 8. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.


Figure 5AhCreERCatnblox ( ex3 ) / + compared to AhCreER Catnblox ( ex3 ) / + Cdh1fl / + at day 10 post - induction . Note the presence of colonic lesions in the colon of AhCreER Catnblox ( ex3 ) / + Cdh1fl / + mice ( arrows ) . Scale bar , 100 μm ; red bar indicates proliferative zone ( BrdU ) . Scoring of BrdU - positive cells per half - crypt in the small intestine of wild - type , AhCreERCdh1fl / + , AhCreERCatnblox ( ex3 ) / + and AhCreERCatnblox ( ex3 ) / + Cdh1fl / + mice at day 10 post - induction . N ≥ 3 , at least 25 crypts per mouse were scored . There was significantly higher proliferation in the AhCreERCatnblox ( ex3 ) / + Cdh1fl / + mice ( P = 0 . 028 , one - sided Mann - Whitney U - test ) . In vitro growth of crypts ( small intestine ) from mice as indicated at day 5 post - induction without R - spo1 . Quantification of sphere - forming efficiency of AhCreERCatnblox ( ex3 ) / + AhCreERCatnblox ( ex3 ) / + Cdh1fl / + , day 5 post - induction . N = 3 mice per genotype , mean of 6 technical replicates per mouse , P = 0 . 04 one - sided Mann - Whitney U - test . Survival of Lgr5CreERCatnblox ( ex3 ) / + and Lgr5CreERCatnblox ( ex3 ) / + Cdh1fl / + shows significant acceleration ( P = 0 . 000123 , log - rank test ) after E - cadherin reduction . About 85 % ( 6 / 7 mice ) had colonic lesions , as identified with β - cateninIHC in contrast to Lgr5CreERCatnblox ( ex3 ) / + mice .

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Figure 5AhCreERCatnblox(ex3)/+ compared to AhCreER Catnblox(ex3)/+ Cdh1fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCreER Catnblox(ex3)/+ Cdh1fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU).Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCreERCdh1fl/+, AhCreERCatnblox(ex3)/+ and AhCreERCatnblox(ex3)/+Cdh1fl/+mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCreERCatnblox(ex3)/+Cdh1fl/+mice (P = 0.028, one-sided Mann-Whitney U-test).In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCreERCatnblox(ex3)/+AhCreERCatnblox(ex3)/+Cdh1fl/+, day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann-Whitney U-test.Survival of Lgr5CreERCatnblox(ex3)/+ and Lgr5CreERCatnblox(ex3)/+Cdh1fl/+ shows significant acceleration (P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-cateninIHC in contrast to Lgr5CreERCatnblox(ex3)/+mice.


Figure 6Proximity ligation assay for E - cadherin : β - catenin on a tissue microarray of SPT patients . PLA dots / cells in normal and tumour tissue were counted . Each dot in the boxplot represents the average for a single patient . Staining for β - catenin confirms accumulation of nuclear β - catenin in tumour tissue . Representative pictures are shown . N = 18 , statistics : Mann - Whitney U - test , P = 0 . 0009051 . White scale bar , 50 μm . Correlation of several Wnt target genes with the expression of E - cadherin ( CDH1 ) was analysed in 269 HCC patients ( TCGA provisional ) , and linear regression line ( blue ) and confidence region ( shaded ) were added .

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Figure 6Proximity ligation assay for E-cadherin:β-catenin on a tissue microarray of SPT patients. PLA dots/cells in normal and tumour tissue were counted. Each dot in the boxplot represents the average for a single patient. Staining for β-catenin confirms accumulation of nuclear β-catenin in tumour tissue. Representative pictures are shown. N = 18, statistics: Mann-Whitney U-test, P = 0.0009051. White scale bar, 50 μm.Correlation of several Wnt target genes with the expression of E-cadherin (CDH1) was analysed in 269 HCC patients (TCGA provisional), and linear regression line (blue) and confidence region (shaded) were added.


Figure 1 ( A ) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues ( T ) and adjacent nontumor tissues ( N ) . The graph represents CRIP1 protein expression normalized to GAPDH levels ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , paired ( A , Student ' s t - test ( B ) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues . Scale bars : 20 μm . Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , paired B ) Student ' s t - test ( C ) CRIP1 mRNA expression in the TCGA - LIHC dataset . ( D ) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , unpaired Student ' s t - testThe protein ( F ) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes . ( G ) Relative CRIP1 protein level compared to normal LO2 hepatocytes . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , unpaired G ) Student ' s t - test ( H - I ) Kaplan - Meier survival analyses of OS ( H ) and PFS ( I ) based on CRIP1 expression levels in 108 HCC patients . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using log - rank test ( H - I ) .

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Figure 1(A) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues (T) and adjacent nontumor tissues (N). The graph represents CRIP1 protein expression normalized to GAPDH levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired (A, Student's t-test(B) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues. Scale bars: 20 μm. Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired B) Student's t-test(C) CRIP1 mRNA expression in the TCGA-LIHC dataset. (D) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired Student's t-testThe protein (F) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes. (G) Relative CRIP1 protein level compared to normal LO2 hepatocytes. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired G) Student's t-test(H-I) Kaplan-Meier survival analyses of OS (H) and PFS (I) based on CRIP1 expression levels in 108 HCC patients. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using log-rank test (H-I).


Figure 2 ( B ) Tumor sphere formation assay in CRIP1 - overexpressing Huh7 and Hep3B cells . The data are shown as the relative fold change spheres compared with the vector group ( right ) . Scale bar : 50 μm . Data information : Data represent the mean ± SD at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test ( B , ( C ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - overexpressing Huh7 and Hep3B cells . ( H ) Tumor sphere formation assay in CRIP1 - knockdown MHCC - 97H and HepG2 cells . The data are shown as the relative fold change spheres compared with the siCtrl group ( right ) . Scale bar : 50 μm . Data information : Data represent the mean ± SD at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test H , ( I ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - knockdown MHCC - 97H and HepG2 cells .

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Figure 2(B) Tumor sphere formation assay in CRIP1-overexpressing Huh7 and Hep3B cells. The data are shown as the relative fold change spheres compared with the vector group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (B,(C) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 and Hep3B cells.(H) Tumor sphere formation assay in CRIP1-knockdown MHCC-97H and HepG2 cells. The data are shown as the relative fold change spheres compared with the siCtrl group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test H,(I) Western blot analysis of HCC cancer stem cell markers in CRIP1-knockdown MHCC-97H and HepG2 cells.


Figure 3 ( G ) Western blot analysis of cancer stem cell markers in the indicated groups .

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Figure 3(G) Western blot analysis of cancer stem cell markers in the indicated groups.


Figure 4 ( A ) Western blot analysis of BBOX1 protein levels in CRIP1 - overexpressing Huh7 and Hep3B cells . ( B ) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC - 97H and HepG2 cells . ( D ) Western blot analysis of BBOX1 in CRIP1 - overexpressing Huh7 cells treated with CHX ( 100 μg / ml ) for the indicated durations ( lfte ) . The graph shows quantification of relative BBOX1 levels ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 and * * P < 0 . 01 . Differences were tested using an unpaired 2 - tailed Student ' s t - test D ) . ( E ) Western blot analysis of BBOX1 protein levels in CRIP1 - overexpressing Huh7 and Hep3B cells treated with MG132 ( 10 μM ) for 10 h . D ) . ( F ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 and Hep3B cells . ( G ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs . ( H ) Co - immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1 , STUB1 , and BBOX1 in Huh7 and Hep3B cells . ( I ) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1 . ( J ) Co - immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1 - Myc and CRIP1 siRNAs .

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Figure 4(A) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells. (B) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC-97H and HepG2 cells.(D) Western blot analysis of BBOX1 in CRIP1-overexpressing Huh7 cells treated with CHX (100 μg/ml) for the indicated durations (lfte). The graph shows quantification of relative BBOX1 levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01. Differences were tested using an unpaired 2-tailed Student's t-test D).(E) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells treated with MG132 (10 μM) for 10 h. D).(F) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells.(G) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs.(H) Co-immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1, STUB1, and BBOX1 in Huh7 and Hep3B cells.(I) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1.(J) Co-immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1-Myc and CRIP1 siRNAs.


Figure 5 ( B ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant . ( C ) Western blot analysis of BBOX1 - WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1 . tumor sphere formation ability and quantification of spheres ( E ) , in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant . Scale bar : 50 μm . Data information : Data represent the mean ± SD of at least three independent experiments . * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - testprotein levels of HCC cancer stem cell markers ( G ) in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant .

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Figure 5(B) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.(C) Western blot analysis of BBOX1-WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1.tumor sphere formation ability and quantification of spheres (E), in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-testprotein levels of HCC cancer stem cell markers (G) in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.


Figure 6Representative images of tumor sphere formation and quantification of spheres ( C ) in CRIP1 - overexpressing Huh7 cells with or without iCRT3 ( 50 μM ) for 24 h . Scale bar : 50 μm . Data information : Data represent the mean ± SD of at least three independent experiments . * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test ( E ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - overexpressing Huh7 cells with or without iCRT3 ( 50 μM ) for 24 h . ( F ) Coimmunoprecipitation and immunoblot analyses of β - catenin acetylation in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) for 24 h . ( G ) Western blot analysis of cytoplasmic and nuclear fractions of β - catenin in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) for 24h . ( I ) Co - immunoprecipitation and immunoblot analyses of β - catenin ubiquitination in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) and EX527 ( 20 μM ) for 24 h .

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Figure 6Representative images of tumor sphere formation and quantification of spheres (C) in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P <0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test(E) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h.(F) Coimmunoprecipitation and immunoblot analyses of β-catenin acetylation in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24 h.(G) Western blot analysis of cytoplasmic and nuclear fractions of β-catenin in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24h.(I) Co-immunoprecipitation and immunoblot analyses of β-catenin ubiquitination in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) and EX527 (20 μM) for 24 h.


Figure 7 ( A ) IHC analysis of CRIP1 , BBOX1 , β - catenin , CD133 , OCT4 , and NANOG using TAM ( left ) . Scale bars : 20 μm . Correlation analysis of CRIP1 expression with BBOX1 , β - catenin , CD133 , OCT4 , and NANOG expression in HCC tissues ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . . * * P < 0 . 01 and * * * P < 0 . 001 . Chi - square test was performed to determine the correlation ( A ) .

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Figure 7(A) IHC analysis of CRIP1, BBOX1, β-catenin, CD133, OCT4, and NANOG using TAM (left). Scale bars: 20 μm. Correlation analysis of CRIP1 expression with BBOX1, β-catenin, CD133, OCT4, and NANOG expression in HCC tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments.. **P <0.01 and ***P < 0.001. Chi-square test was performed to determine the correlation (A).


Figure 1Heatmaps of AIB1 - YAP co - activated , YAP activated , AIB1 - YAP corepressed , and YAP repressed genes shown in panel ( A ) . Red and blue indicate high and low levels , respectively . RNA - sequencing was performed in triplicate and statistics in Panel ( A - B ) were calculated by EdgeR with Benjamini - Hochberg multiple test correction in R . Select genes are regulated by YAP and AIB1 , others are YAP regulated independently of AIB1 . Endogenous gene expression changes assessed by RT - qPCR . Significant genomic co - occupancy between AIB1 and TEAD in normal breast and breast cancer cells . Overlap of AIB1 and TEAD4 ChIP - seq peaks in MCF10A and MCF7 cell lines . Select peaks from MCF10A ChIP - seq shown . ChIP - sequencing performed in triplicate . TEAD binding motifs are enriched within AIB1 occupied sites across the genome . HOMER de novo motif analysis of AIB1 ChIP - seq peaks in MCF10A and MCF7 cells . AIB1 modulates TEAD transcriptional activity in a YAP dependent manner . HEK293T cells were transfected with CTGF - promoter reporter and expression vectors of indicated proteins . AIB1 is engaged with TEAD on co - activated target promoters in normal cells . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A cells . Levels of precipitated chromatin assessed by qPCR . AIB1 is engaged with TEAD on co - activated target promoters in early stage breast cancer cells . Sequential ChIP - reChIP performed with indicated antibodies in MCFDCIS cells . Levels of precipitated chromatin assessed by qPCR .

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Figure 1Heatmaps of AIB1-YAP co-activated, YAP activated, AIB1-YAP corepressed, and YAP repressed genes shown in panel (A). Red and blue indicate high and low levels, respectively. RNA-sequencing was performed in triplicate and statistics in Panel (A-B) were calculated by EdgeR with Benjamini-Hochberg multiple test correction in R.Select genes are regulated by YAP and AIB1, others are YAP regulated independently of AIB1. Endogenous gene expression changes assessed by RT-qPCR.Significant genomic co-occupancy between AIB1 and TEAD in normal breast and breast cancer cells. Overlap of AIB1 and TEAD4 ChIP-seq peaks in MCF10A and MCF7 cell lines. Select peaks from MCF10A ChIP-seq shown. ChIP-sequencing performed in triplicate.TEAD binding motifs are enriched within AIB1 occupied sites across the genome. HOMER de novo motif analysis of AIB1 ChIP-seq peaks in MCF10A and MCF7 cells.AIB1 modulates TEAD transcriptional activity in a YAP dependent manner. HEK293T cells were transfected with CTGF-promoter reporter and expression vectors of indicated proteins.AIB1 is engaged with TEAD on co-activated target promoters in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.AIB1 is engaged with TEAD on co-activated target promoters in early stage breast cancer cells. Sequential ChIP-reChIP performed with indicated antibodies in MCFDCIS cells. Levels of precipitated chromatin assessed by qPCR.


Figure 2ANCO1 is recruited to YAP - TEAD by AIB1 . Positive Proximity Ligation Assay ( PLA ) signal ( white foci , falsely colored 594nm signal ) shown between ANCO1 - TEAD , ANCO1 - YAP , AIB1 - TEAD , and AIB1 - YAP , in control cells are lost in AIB1 knockdown . Representative images of Proximity Ligation Assay ( PLA ) between indicated proteins in MCF10A shGFP ( control ) and shAIB1 cell lines using antibodies ( species ) as indicated ; Scale bars shown indicate 25μm . YAP overexpression represses , AIB1 knockdown dysregulates , and ANCO1 knockdown amplifies genes at the 1q21 . 3 locus in MCF10A and MCFDCIS cells . Gene set enrichment analysis of RNA - seq data and cDNA array data generated from MCF10A cell lines as indicated . YAP represses 1q21 . 3 localized genes . Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP / S94A ; levels were assessed by RT - qPCR . ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells ; levels were assessed by RT - qPCR . YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown . Levels of endogenous 1q21 . 3 genes were assessed in YAP or S94A - expressing , control or shANCO1 MCF10A cells by RT - qPCR . TEAD is engaged at annotated enhancer regions within 1q21 . 3 . Analysis of TEAD4 ChIP - seq in MDA - MB - 231 ( GSE66081 ) , MCF7 , and A549 Cells ( GSE32465 ) at 1q21 . 3 with enhancers scored by GeneHancer . Enhancer regions of interest indicated between dotted lines . TEAD , YAP , AIB1 , and ANCO1 are engaged at annotated enhancer regions within 1q21 . 3 . Chromatin Immunoprecipitation ( ChIP ) performed with indicated antibodies in MCF10A cells . AIB1 is engaged with TEAD on superenhancer sites at 1q21 . 3 in normal cells . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A cells . Levels of precipitated chromatin assessed by qPCR . ANCO1 is recruited to superenhancers at 1q21 . 3 by AIB1 and is significantly reduced at 1q21 . 3 following AIB1 knockdown . ChIP performed with ANCO1 antibodies in control or shAIB1 cells . Levels of precipitated chromatin assessed by qPCR .

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Figure 2ANCO1 is recruited to YAP-TEAD by AIB1. Positive Proximity Ligation Assay (PLA) signal (white foci, falsely colored 594nm signal) shown between ANCO1-TEAD, ANCO1-YAP, AIB1-TEAD, and AIB1-YAP, in control cells are lost in AIB1 knockdown. Representative images of Proximity Ligation Assay (PLA) between indicated proteins in MCF10A shGFP (control) and shAIB1 cell lines using antibodies (species) as indicated; Scale bars shown indicate 25μm.YAP overexpression represses, AIB1 knockdown dysregulates, and ANCO1 knockdown amplifies genes at the 1q21.3 locus in MCF10A and MCFDCIS cells. Gene set enrichment analysis of RNA-seq data and cDNA array data generated from MCF10A cell lines as indicated.YAP represses 1q21.3 localized genes. Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP/S94A; levels were assessed by RT-qPCR.ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells; levels were assessed by RT-qPCR.YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown. Levels of endogenous 1q21.3 genes were assessed in YAP or S94A-expressing, control or shANCO1 MCF10A cells by RT-qPCR.TEAD is engaged at annotated enhancer regions within 1q21.3. Analysis of TEAD4 ChIP-seq in MDA-MB-231(GSE66081), MCF7, and A549 Cells (GSE32465) at 1q21.3 with enhancers scored by GeneHancer. Enhancer regions of interest indicated between dotted lines.TEAD, YAP, AIB1, and ANCO1 are engaged at annotated enhancer regions within 1q21.3. Chromatin Immunoprecipitation (ChIP) performed with indicated antibodies in MCF10A cells.AIB1 is engaged with TEAD on superenhancer sites at 1q21.3 in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.ANCO1 is recruited to superenhancers at 1q21.3 by AIB1 and is significantly reduced at 1q21.3 following AIB1 knockdown. ChIP performed with ANCO1 antibodies in control or shAIB1 cells. Levels of precipitated chromatin assessed by qPCR.


Figure 3YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown , but further enhanced by ANCO1 knockdown . Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days . 10x and 40x magnifications shown , Scale bar = 200μm . Quantification of aberrant spheres per field displayed in panel ( A ) , n = number of spheres . Quantification of size ( arbitrary units ) of aberrant spheres per field displayed in panel ( A ) , relative to control YAP S94A shGFP spheres . n = number of spheres . MCFDCIS xenograft tumors have increased expression of AIB1 - YAP , EMT , and 1q21 . 3 genes during the invasive transition . Schematic of experiment shown . Gene set enrichment analysis of RNA - seq data generated from MCFDCIS xenografts at indicated time points . Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions . Representative images of H & E , p63 , and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions . Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions . 20x magnification ; Scale bar = 100μm . Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel ( E ) .

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Figure 3YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown, but further enhanced by ANCO1 knockdown. Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days. 10x and 40x magnifications shown, Scale bar = 200μm.Quantification of aberrant spheres per field displayed in panel (A), n= number of spheres.Quantification of size (arbitrary units) of aberrant spheres per field displayed in panel (A), relative to control YAP S94A shGFP spheres. n= number of spheres.MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions. Representative images of H&E, p63, and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions. Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions. 20x magnification; Scale bar = 100μm. Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel (E).


Figure 4ANCO1 levels decrease in breast cancer . ANCO1 mRNA expression from normal tissue ( GTEX ) and Tumor ( TCGA ) for breast samples . Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions . DCIS lesions can be stratified to either ANCO1 negative ( center panel ) , ANCO1 positive ( right panel ) . Scale bar = 50μm . High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients . KM plot showing progression free survival in patients with stratified ANCO1 expression . n = 360 patients . Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21 . 3 genes , without altered AIB1 - YAP co - activated gene expression . Dot plots showing TCGA Pan - Cancer data accessed via Xenabroswer . Patients were stratified to high or low expression of ANCO1 by median expression .

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Figure 4ANCO1 levels decrease in breast cancer. ANCO1 mRNA expression from normal tissue (GTEX) and Tumor (TCGA) for breast samples.Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions. DCIS lesions can be stratified to either ANCO1 negative (center panel), ANCO1 positive (right panel). Scale bar = 50μm.High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients. KM plot showing progression free survival in patients with stratified ANCO1 expression. n= 360 patients.Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21.3 genes, without altered AIB1-YAP co-activated gene expression. Dot plots showing TCGA Pan-Cancer data accessed via Xenabroswer. Patients were stratified to high or low expression of ANCO1 by median expression.


Figure 1 ( C ) Flag - tagged Raptor was transfected with HA - tagged ArfGAP1 or HA - tagged RFP1 in wildtype ( WT ) or RagA / B knockout ( KO ) HEK293A cells for 24 hours . Cell lysates were immunoprecipitated ( IP ) with anti - Flag beads . IP or WCL samples were probed for HA - tagged ArfGAP1 or HA - tagged RFP1 , Flag - tagged Raptor , RagA , RagB , and Vinculin the loading control . ( G ) RagA / B KO HEK293A cells were transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) for 72 hours . Cell lysates were immunoprecipitated ( IP ) with anti - HA beads . IP or WCL samples were probed for HA and mTORC1 components mTOR , Raptor and mLST8 . Vinculin was probed as a control .

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Figure 1(C) Flag-tagged Raptor was transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 in wildtype (WT) or RagA/B knockout (KO) HEK293A cells for 24 hours. Cell lysates were immunoprecipitated (IP) with anti-Flag beads. IP or WCL samples were probed for HA-tagged ArfGAP1 or HA-tagged RFP1, Flag-tagged Raptor, RagA, RagB, and Vinculin the loading control.(G) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) for 72 hours. Cell lysates were immunoprecipitated (IP) with anti-HA beads. IP or WCL samples were probed for HA and mTORC1 components mTOR, Raptor and mLST8. Vinculin was probed as a control.


Figure 2 ( D ) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 ( shArfGAP1 ) or GFP ( shGFP ) control . Cells were cultured under normal condition ( NC ; lanes 1 and 7 ) , or starved of amino acids for 1 hour ( - AA 1h ; lanes 2 and 8 ) , followed by either maintaining in amino acid starvation media ( - AA ; lanes 3 and 9 ) , or addition of 1 mM of Leu ( + Leu ; lanes 4 and 10 ) , Gln ( + Gln ; lanes 5 and 11 ) , or Asn ( + Asn ; lanes 6 and 12 ) for 2 hours . mTORC1 activity was analyzed similar to ( B ) . Protein levels of ArfGAP1 were confirmed by immunoblotting . S6K1 and Actin were probed as controls . ( E ) RagA / B KO HEK293A cells were co - transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) with Myc - tagged S6K1 . Cells were starved of amino acids ( - AA ) for 2 hours and then 4mM of Gln or Asn were added for 2 hours . mTORC1 was analyzed similar to ( B ) . IP or WCL samples were probed for HA , Myc , and phosphorylated S6K1 at Thr 389 . Actin serve as a loading control .

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Figure 2(D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls.(E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control.


Figure 3 ( B ) Fluorescence Lifetime Imaging Microscopy ( FLIM ) and Förster resonance energy transfer ( FRET ) analysis were performed in wildtype ( WT , left ) or RagA / B KO ( middle ) HAP1 cells expressing mCherry - tagged ArfGAP1 and endogenously GFP - tagged Raptor . Cells either had amino acids ( + AA ) or were starved of amino acids ( - AA ) . At least 20 cells were analyzed in each group . Box plots shows the percentage of pixels with ≥ 30 % FRET efficiency of co - transfected cells . RFP1 was expressed and analyzed as a negative control . The whiskers of the boxplot represent the minimum and maximum values , while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median . Right - Expression level of RagA / B was confirmed Western blot analysis . Actin was used as a control .

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Figure 3(B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.


Figure 4 ( A ) Mouse embryonic fibroblast ( MEF ) cells were transfected with HA - tagged ArfGAP1 . Immunofluorescence staining was assessed for HA - tagged ArfGAP1 ( blue ) , mTOR ( green ) , and LAMP2 ( red , lysosomal marker ) . Left - A representative example of the immunofluorescence staining ( scale bar , 10 μm ) . The top left image shows the staining of HA - tagged ArfGAP1 , with a white outline highlighting the region that is positive for HA - tagged ArfGAP1 . This outline was overlaid with the mTOR channel ( top right ) and the LAMP2 channel ( bottom left ) . The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm ( Rizk et al . , 2014 ; bottom right ) . The co - localization of mTOR and LAMP2 was analyzed for the region within the outline ( HA - ArfGAP1 positive ) and the region outside of the outline ( HA - ArfGAP1 - negative ) , respectively . Right - In each microscopy field , the image was processed as described above , and the co - localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm ( Rizk et al . , 2014 ) . The quantification was performed for the subgroups of cells that are positively or negatively stained for HA - tagged ArfGAP1 , respectively , and compared using Student ' s t - test . Columns represent Mean ± SEM ( N = 3 ) . ( D ) MIA Paca - 2 cells stably expressing FLAG - tagged or HA - tagged TMEM192 cells were cultured under normal condition ( NC ) or starved of amino acids ( - AA ) , and then stimulated with 2 mM glutamine and asparagine ( + Gln / Asn ) for 2 hours . HA - tagged TMEM192 was immunoprecipitated ( IP ) with HA beads and IP and whole cell lysates ( WCL ) were probed for ArfGAP1 , HA , RagA , or p18 ( LAMTOR1 ) . Flag , pS6K1 ( mTORC1 activity ) , S6K1 , and Vinculin were probed for as controls .

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Figure 4(A) Mouse embryonic fibroblast (MEF) cells were transfected with HA-tagged ArfGAP1. Immunofluorescence staining was assessed for HA-tagged ArfGAP1 (blue), mTOR (green), and LAMP2 (red, lysosomal marker). Left- A representative example of the immunofluorescence staining (scale bar, 10 μm). The top left image shows the staining of HA-tagged ArfGAP1, with a white outline highlighting the region that is positive for HA-tagged ArfGAP1. This outline was overlaid with the mTOR channel (top right) and the LAMP2 channel (bottom left). The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm (Rizk et al., 2014; bottom right). The co-localization of mTOR and LAMP2 was analyzed for the region within the outline (HA-ArfGAP1 positive) and the region outside of the outline (HA-ArfGAP1-negative), respectively. Right- In each microscopy field, the image was processed as described above, and the co-localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm (Rizk et al., 2014). The quantification was performed for the subgroups of cells that are positively or negatively stained for HA-tagged ArfGAP1, respectively, and compared using Student's t-test. Columns represent Mean ± SEM (N = 3).(D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls.


Figure 5 ( D ) Top - Input of GST , GST - tagged ArfGAP1 , and GST - tagged ArfGAP1 L207D / V279D that was us in the in vitro binding assays . Bottom - Flag - tagged mTOR , Flag - tagged Raptor , and Flag - tagged mLST8 were overexpressed in HEK293A cells . Cell lysates were immunoprecipitated ( IP ) with anti - Flag beads and incubated with either GST ( control ) , GST - tagged ArfGAP1 , or GST - ArfGAP1 L207D / V279D . IP or WCL samples were probed for Flag or GST .

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Figure 5(D) Top - Input of GST, GST-tagged ArfGAP1, and GST-tagged ArfGAP1 L207D/V279D that was us in the in vitro binding assays. Bottom - Flag-tagged mTOR, Flag-tagged Raptor, and Flag-tagged mLST8 were overexpressed in HEK293A cells. Cell lysates were immunoprecipitated (IP) with anti-Flag beads and incubated with either GST (control), GST-tagged ArfGAP1, or GST-ArfGAP1 L207D/V279D. IP or WCL samples were probed for Flag or GST.


Figure 6 ( A ) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression . Overall survival and disease - free survival were compared between these two groups , as shown in Kaplan - Meier curves . Median survivals ( in months ) , log - rank P values , and hazard ratios ( HR ) with 95 % confidence intervals are indicated . ( D ) MIA Paca - 2 cells stably expressing short hairpin RNAs ( shRNAs ) against ArfGAP1 or GFP ( control ) were plated and treated with mTOR inhibitor Torin 1 ( 250 nM ) or vehicle control for 48 hours prior to cell counting . Columns represent Mean ± SEM ( N = 6 ) . Student ' s t - test shGFP Vehicle vs . shGFP Torin 1 ( P = 2 . 83 E - 07 ) , shGFP Vehicle vs . shArfGAP1 Vehicle ( * P = 0 . 025 ) , shGFP Torin 1 vs , shArfGAP1 Torin 1 ( P = 0 . 139 ) , and shArfGAP1 Vehicle vs . shArfGAP1 Torin 1 ( P = 1 . 22 E - 07 ) . " ns " stands for " not significant " .

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Figure 6(A) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression. Overall survival and disease-free survival were compared between these two groups, as shown in Kaplan-Meier curves. Median survivals (in months), log-rank P values, and hazard ratios (HR) with 95% confidence intervals are indicated.(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".


Figure 1 ( B ) Immunoblot showing CENP - AEA protein level at the indicated time in RPE - 1 cells . ( C ) Representative immunofluorescence images showing CENP - A reloading at CENP - B - marked centromeres in DLD - 1 cells . White dashed circles contour nuclei . Scale bar , 5 µm . ( D ) Quantification of the percentage of cells showing centromeric CENP - A 24 hr or 48 hr after IAA WO . Each dot represents one experiment ( ~ 30 - 50 cells per condition per experiment ) , error bars represent standard deviation ( SD ) of 5 independent experiments . ( E ) Quantification of centromeric CENP - A levels normalized to non - treated level . Each dot represents one experiment , error bars represent SD . Unpaired t test : * * * p = 0 . 0005 . ( F ) Stills of live cell imaging to follow de novo CENP - AEA reloading in RPE - 1 cells harboring endogenously tagged CENP - Bmcherry . Images were taken every 15 min . White dashed circles contour nuclei prior / after mitosis and cells during mitosis based on bright field images . ( G ) Dot plot showing the timing of CENP - AEA reloading after anaphase onset in the indicated cell lines . Each dot represents one cell , error bars represent standard deviation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells.(C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm.(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.(E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005.(F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images.(G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation.


Figure 2 ( B ) Full coverage plot of chromosome 17 centromeric array ( chr17 : 22 , 500 , 000 - 26 , 900 , 000 of the hg38 assembly ) showing enrichment of CENP - A by CUT & RUN - seq in the untreated ( NT ) and IAA wash out ( WO ) sample . CENP - A is reloaded to cen17 _ 1 ( D17Z1 ) . A shift of CENP - A occupancy to cen17 _ 2 ( D17Z1B ) is not detected . ( F ) Representative images showing ectopic CENP - A on a chromosome spread after induction of CENP - A overexpression in DLD - 1 cells . Centromere position is marked using immunofluorescence staining for CENP - B . Yellow dashed lines contour representative chromosomes . Scale bar , 2µm . Schematic to the right illustrate the observed CENP - AmRFP - DHFR localization pattern on a chromosome . Right panel : Comparison of CENP - ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX / TMP treatment . Each dot represents one centromere or a region on the chromosome arms . ( G ) Schematic illustration for the experiments shown in H , I . ( H ) Representative images of chromosome spreads after re - expression of endogenous CENP - AEA into a CENP - AmRFP - DHFR overexpression background and subsequent removal of the overexpression as illustrated in ( G ) . Antibody against CENP - C was used to score for the presence of the functional centromere . Scale bar , 5µm . ( I ) Quantification of the indicated events after one CENP - AOFF / ON cycle compared to treatment shown in ( G ) .

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Figure 2(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.(G) Schematic illustration for the experiments shown in H, I. (H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm. (I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).


Figure 3 ( A ) Schematic illustration of the CENP - AOFF / ON cycle performed in the experiments shown in B , C . ( B ) Representative images of de novo CENP - A reloading in CENP - B wild - type ( + / + ) and CENP - B knock - out ( - / - ) cells . Cells with centromeric CENP - A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP - A . Scale bar , 5 µm . ( C ) Quantification of relative number of RPE - 1 cells with centromeric CENP - A in the indicated conditions . Each dot represents one experiment with at least 20 cells per condition . Error bars represent standard error of the mean ( SEM ) from 3 independent experiments . ( E ) Representative images of DLD - 1 CENP - B ( + / + ) or ( - / - ) cells in late M phase following one CENP - AOFF / ON cycle . EdU staining was used to confirm successful wash - out of Palbociclib and cell progressing through S - phase . Yellow dashed lines contour nucleus of cells in late M phase . Scale bar 5 µm . ( F ) Quantification of indicated events observed in late M phase cells in the indicated cell lines . Error bars show SEM from 4 independent experiments .

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Figure 3(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C. (B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm. (C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments. (E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.


Figure 4 ( C ) Representative IF - FISH images of mitotic spreads in non - treated cell ( NT ) and following a CENP - AOFF / ON cycle ( IAA WO ) . Chromosomes are contoured by a green ( X ) , magenta ( Y ) or white ( other autosomes ) dashed line . The centromeres of chromosome X / Y are highlighted by a green / magenta arrow . Scale bar , 2 µm . ( D ) Quantification of CENP - C ( as read - out of CENP - A ) presence at the X ( green ) or Y ( magenta ) centromere in the indicated cell lines . Each dot represents one experiment . Error bars represent SD from 3 independent experiments . Unpaired t test , * * p = 0 . 0042 . ( G ) Representative IF - FISH images showing CENP - C localization on the Y chromosome in cell growing under selective pressure ( G418 ) following a CENP - AOFF / ON cycle . Chromosomes are highlighted by a white dashed contour line . CENP - C was found at the native centromere position ( top panel , blue ) , the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr - Y painting probe staining ( second panel , orange ) or CENP - C was found on a different location on the Y chromosome and did not overlap with Cen - Y DNA ( lower panels , red ) . Scale bar , 2 µm . ( H ) Quantification of indicated events depicted in G . Each dot represents one experiment ( > 20 spreads for experiment ) . Error bars represent SEM from 3 independent experiments . Unpaired t - test , * p = 0 . 0137 and 0 . 039 .

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Figure 4(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.(D) Quantification of CENP-C (as read-out of CENP-A) presence at the X (green) or Y (magenta) centromere in the indicated cell lines. Each dot represents one experiment. Error bars represent SD from 3 independent experiments. Unpaired t test, **p=0.0042.(G) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm. (H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039.


Figure 5 ( B ) Representative immunofluorescence images showing CENP - C recruitment at the LacO array in the indicated conditions . LacO array is displayed in the insets . ( C ) Bar plot showing frequency of CENP - C ( orange ) or CENP - A ( green ) recruitment to LacO array in the indicated conditions . Each dot represents one independent experiment ( > 20 cell analyzed for experiment ) . ( D ) Quantification of CENP - C ( orange ) or CENP - A ( green ) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non - treated conditions at the mCherry - LacI - CENP - B - LacO array using CENP - A or CENP - C antibody . Each dot represents one independent experiment ( > 20 cell analyzed for experiment ) . Error bars represent SEM from 3 independent experiments . ( E ) Representative immunofluorescence images showing CENP - A recruitment to ectopic mCherry - LacI - CENP - B in the indicated conditions . LacO array is shown in the inset . ( F ) Quantification of the frequency of CENP - C recruitment to LacO arrays by different LacI constructs . Error bars represent SEM from 5 independent experiments . ( G ) CENP - B immunoblot following GST pulldown experiments using GST tagged CENP - C ( 1 - 727 ) as bait and with the indicated proteins as preys .

O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets.(C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment).(D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments.(E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset.(F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments.(G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.


Figure 6 ( C ) Representative immunofluorescence images showing CENP - A and CENP - C reloading in the indicated conditions . Cells with centromeric CENP - A / CENP - C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP - A . The centromere position is marked using immunofluorescence staining for CENP - B . White dashed lines contour DAPI stained nucleus of representative cells . ( D ) Bar plot showing the percentage of cells with CENP - A and CENP - C co - localization after a CENP - A / COFF / ON cycle in the indicated conditions . Error bars show SD from 5 independent experiments . ( E ) Box plot of CUT & RUN qPCR for CENP - A ( green ) or CENP - C ( orange ) . Box plot shows median , 25th and 75th percentiles , whiskers show minimum and maximum values . Results from 3 independent experiments including combined data from qPCR reactions using alpha - satellite primers and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells.(D) Bar plot showing the percentage of cells with CENP-A and CENP-C co-localization after a CENP-A/COFF/ON cycle in the indicated conditions. Error bars show SD from 5 independent experiments.(E) Box plot of CUT&RUN qPCR for CENP-A (green) or CENP-C (orange). Box plot shows median, 25th and 75th percentiles, whiskers show minimum and maximum values. Results from 3 independent experiments including combined data from qPCR reactions using alpha-satellite primers and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


Figure 7 ( C ) Representative immunofluorescence images showing exogenous CENP - AmRFP - DHFR reloading in the presence ( control ) , but not in the absence ( sample ) of endogenous CENP - A and CENP - C . Cells with centromeric CENP - A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP - A . ( D ) Quantification of relative number of cells showing centromeric eCENP - AmRFP - DHFR in the indicated conditions . Error bars represent SEM from 3 independent experiments . ( E ) Bar plot of CUT & RUN qPCR quantification using CENP - A antibody and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats . ( F ) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP - CmRFP - DHFR in the presence ( control ) and in the absence ( sample ) of endogenous CENP - A and CENP - C . Cells were arrested with Colcemid for 3 h prior to spread . Cells with centromeric CENP - C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP - C . Chromosomes in inset are highlighted using a white dashed line . ( G ) Quantification of relative number of cells with eCENP - CmRFP - DHFR in the indicated conditions . Each dot represents one independent experiment with at least 30 cells per condition . ( H ) Bar plot of CUT & RUN qPCR results using CENP - C antibody and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats . Each dot represents one independent experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A. (D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments.(E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.(F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. (G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition.(H) Bar plot of CUT&RUN qPCR results using CENP-C antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Each dot represents one independent experiment.


Figure 8 ( A ) Gating strategy for the sorting of freshly purified resting human CD4 + T cells based on CENP - A expression . Dead cells were excluded ( first plot ) using Fixable Viability Dye eFluor 780 , CENP - Ahigh and CENP - Alow cells ( both CENP - B + ) were sorted ( second plot ) . Post - sort analysis of each population is then shown ( third and fourth plots ) . ( B ) Representative images of sorted CD4 + T cells with high or low CENP - A level . The nucleus is contoured by a white dashed line . Scale bar , 5 µm . ( D ) Tracking of cell division by CFSE dilution . The black line separates cells that have not divided ( nº of div = 0 , CFSE - high ) from those that have undergone at least one division ( nº of div ≥ 1 , CFSE - low ) . ( E ) CENP - A expression and CFSE dilution . Black lines were set based on CENP - A isotype control and CFSE maximal staining at day 0 ( cells in shaded quadrants are CFSE - high / CENP - A - low ) . One representative donor is depicted . ( F ) Absolute number of CFSE - high / CENP - A - low CD4 + T cells ( shaded gate in E ) . Symbols represent individual donors . Error bars represent SEM from 6 independent donors . One - way ANOVA , multiple comparisons , n = 6 . * p < 0 . 05 ; * * p < 0 . 01 . ( G ) Frequency of cells without centromeric CENP - A determined by immunofluorescence microscopy before and post activation . Symbols represent individual donors , n = 4 . Error bars represent SEM from 6 independent donors .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots).(B) Representative images of sorted CD4+ T cells with high or low CENP-A level. The nucleus is contoured by a white dashed line. Scale bar, 5 µm.(D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low). (E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted. (F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01. (G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors.


Figure 1Representative Western blots showing GAPDH association with mitochondria - enriched fractions in different HD models . The levels of GAPDH were normalized to mitochondrial matrix protein , aconitase . n = 3 . * P = 0 . 0005 ; * * P = 0 . 019 ; # P = 0 . 042 . Measurement of GAPDH activity in the mitochondria - enriched fraction isolated from PC12 cells . n = 3 . * P = 0 . 0005 . Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria . The levels of oxidized GAPDH were normalized to beta - actin ( total lysate ) and aconitase ( mitochondrial fraction ) , respectively . n = 2 . * P = 0 . 023 ; * * P = 0 . 039 . Western blot showing co - immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent ( Triton X - 100 ) . Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial ( VDAC and Tom20 ) , endoplasmic reticulum ( calnexin ) , and lysosomal markers ( LAMP1 ) . ctr : control . n = 2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative Western blots showing GAPDH association with mitochondria-enriched fractions in different HD models. The levels of GAPDH were normalized to mitochondrial matrix protein, aconitase. n = 3. *P = 0.0005; **P = 0.019; #P = 0.042.Measurement of GAPDH activity in the mitochondria-enriched fraction isolated from PC12 cells. n = 3. *P = 0.0005.Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria. The levels of oxidized GAPDH were normalized to beta-actin (total lysate) and aconitase (mitochondrial fraction), respectively. n = 2. *P = 0.023; **P = 0.039.Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.


Figure 2Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 ( top panel , left ) and Q74 ( bottom panel , left ) and in brain tissues from wild - type ( WT ) ( top panel , right ) and HD transgenic mice ( bottom panel , right ) . An indicated portion of each image ( in yellow ) is expanded to illustrate the mitochondrial morphology . Scale bars : 1 μm . Measurement of mitochondrial ROS production ( using Mitosox ) in different HD models . Results are presented as percent of control [ Q23 , normal fibroblast ( Nor ) , Q7 ] . n = 3 . * P = 0 . 001 ; * * P = 0 . 0001 ; * * * P = 0 . 0001 ; # P = 0 . 0045 . Mitochondrial membrane potential measured by using JC - 1 dye in models of HD and control cells ( MMP , ΔΨm ) . n = 3 . * P = 0 . 008 ; * * P = 0 . 044 ; * * * P = 0 . 025 ; # P = 0 . 005 . Mitochondrial respiratory function in PC12 cells , represented by respiratory control ratio ( RCR ; [ state3 ] / [ state4 ] ) . n = 2 . * P = 0 . 028 . ATP levels in HD patient - derived fibroblast cells . n = 2 . * P = 0 . 003 ; * * P = 0 . 004 . Western blots showing cytochrome c release from mitochondria . n = 2 . * P = 0 . 003 ; * * P = 0 . 003 ; * * * P = 0 . 01 ; # P = 0 . 008 . Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models . n = 3 . * P , * * P , * * * P , # P < 0 . 005 . Representative immunofluorescence of normal and HD patient - derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox . Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson ' s correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS . # P = 0 . 03 ; # # P = 0 . 02 ; * P = 0 . 006 ; * * P = 0 . 005 . Images were acquired at 63 × magnification , and brightness and contrast of images were adjusted by 50 % .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 (top panel, left) and Q74 (bottom panel, left) and in brain tissues from wild-type (WT) (top panel, right) and HD transgenic mice (bottom panel, right). An indicated portion of each image (in yellow) is expanded to illustrate the mitochondrial morphology. Scale bars: 1 μm.Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045.Mitochondrial membrane potential measured by using JC-1 dye in models of HD and control cells (MMP, ΔΨm). n = 3. *P = 0.008; **P = 0.044; ***P = 0.025; #P = 0.005.Mitochondrial respiratory function in PC12 cells, represented by respiratory control ratio (RCR; [state3]/[state4]). n = 2. *P = 0.028.ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004.Western blots showing cytochrome c release from mitochondria. n = 2. *P = 0.003; **P = 0.003; ***P = 0.01; #P = 0.008.Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models. n = 3. *P, **P, ***P, #P < 0.005.Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson's correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.


Figure 3A Representative Western blots showing separation of lysosome - and mitochondria - enriched fractions by a density gradient . Total extracts from different HD models were fractionated by the density gradient . Eight fractions were collected from top ( # 1 ) to the bottom ( # 8 ) . The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient . n = 3 . B , C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild - type ( WT ) and HD transgenic R6 / 2 mice showing mitochondrial ( as indicated by yellow arrows ) association with lysosomal vacuoles ( shown by red arrows ) . The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted . Analysis ( of six independent images ) was carried out by an observer blinded to the experimental conditions . * P < 0 . 023 ; * * P < 0 . 035 . Scale bar : 1 μm . D Western blot showing association of mitochondrial membrane with lysosomal membrane . The presence of membranes of each organelle was assessed using anti - MFN1 , VDAC ( as mitochondria markers ) , and LAMP1 ( as a lysosomal marker ) . n = 3 . E Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , and aconitase ) in the total lysates of PC12 cells with Q23 or Q74 . Mitochondrial protein levels were normalized to beta - actin . n = 3 . * P = 0 . 024 ; * * P = 0 . 0008 ; * * * P = 0 . 03 . F Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , and aconitase ) in the total lysates of normal fibroblast cells and HD patient - derived fibroblasts . Mitochondrial protein levels were normalized to the cytosolic protein , enolase . n = 3 . * P = 0 . 008 ; * * P = 0 . 002 ; # P = 0 . 0008 . G Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , and aconitase ) in the total lysates of mice striatal cells with Q7 and Q111 . n = 3 . * P = 0 . 0008 ; * * P = 0 . 0007 ; # P = 0 . 001 . H Western blot showing mitochondrial contents ( Tom20 and aconitase ) in the total lysates of brain tissues from wild - type ( WT ) and HD transgenic mice . n = 2 . * P = 0 . 02 ; * * P = 0 . 03 * § P = 0 . 01 ; * * § P < 0 . 04 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Representative Western blots showing separation of lysosome- and mitochondria-enriched fractions by a density gradient. Total extracts from different HD models were fractionated by the density gradient. Eight fractions were collected from top (#1) to the bottom (#8). The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient. n = 3.B, C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild-type (WT) and HD transgenic R6/2 mice showing mitochondrial (as indicated by yellow arrows) association with lysosomal vacuoles (shown by red arrows). The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted. Analysis (of six independent images) was carried out by an observer blinded to the experimental conditions. *P < 0.023; **P < 0.035. Scale bar: 1 μm.D Western blot showing association of mitochondrial membrane with lysosomal membrane. The presence of membranes of each organelle was assessed using anti-MFN1, VDAC (as mitochondria markers), and LAMP1 (as a lysosomal marker). n = 3.E Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of PC12 cells with Q23 or Q74. Mitochondrial protein levels were normalized to beta-actin. n = 3. *P = 0.024; **P = 0.0008; ***P = 0.03.F Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of normal fibroblast cells and HD patient-derived fibroblasts. Mitochondrial protein levels were normalized to the cytosolic protein, enolase. n = 3. *P = 0.008; **P = 0.002; #P = 0.0008.G Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of mice striatal cells with Q7 and Q111. n = 3. *P = 0.0008; **P = 0.0007; #P = 0.001.H Western blot showing mitochondrial contents (Tom20 and aconitase) in the total lysates of brain tissues from wild-type (WT) and HD transgenic mice. n = 2. *P = 0.02; **P = 0.03 *§P = 0.01; **§P < 0.04.