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1 Supplementary Figure 1 Dox Cis Cam Pac µmole/l Ub p53 Cytotoxic anticancer agents increase p53 levels but do not generally promote the accumulation of ubiquitinated. Western blots of nuclear extract proteins from ZR751 breast cancer cells treated for 3 h with the indicated concentrations of parthenolide (), doxorubicin (Dox), cisplatin (Cis), camptothecin (Cam), and paclitaxel (Pac). 1
2 Supplementary Figure 2 Ub Ub Ub H159 Jurkat K min min Ub Ub Ub SKOV3.ip1 A375 Ishikawa Ub HEC1B increases the abundance of ubiquitinated in many cancer cells. Western blots of nuclear extracts from head and neck squamous carcinoma (H159), Tcell leukemia (Jurkat), chronic myelogenous leukemia (K562), ovarian carcinoma (SKOV3.ip1), melanoma (A375), and endometrial carcinoma (Ishikawa and HEC1B) cells treated with 15 μmol/l for the times indicated and probed with and poly(adpribose) polymerase () antibodies as indicated (right). The H159 head and neck squamous carcinoma cell line was obtained from Chris Barnes (The University of Texas M. D. Anderson Cancer Center). All other cell lines were from the American Type Culture Collection. 2
3 Supplementary Figure 3 A B MG132 MG132 Ab (hc) Ab (hc) Ab (lc) Ab (lc) IP: control Ab IB: IP: control Ab IB: ubiquitin A nonimmunogenic control antibody does not immunoprecipitate any proteins that crossreact with either or ubiquitinspecific antibodies. A, western blots of proteins immunoprecipitated by a nonimmunogenic control antibody from ZR751 cells treated with 15 μmol/l and/or 10 μmol/l MG132 for 3 h, as indicated, and probed with anti antibodies. B, western blotting as described in A, except that the blot was with antiubiquitin antibodies. 3
4 Supplementary Figure 4 Ubiquitinated accumulation induced by treatment with does not require intact IKK IκBα NFκB or JNK signal transduction pathways. A, western blots of nuclear extract proteins from mouse embryo fibroblasts (MEFs) without (wt) or with homozygous deletion of JNK1 and JNK2 genes treated with 15 μmol/l for the indicated times and probed with antibodies against, p53 and as indicated. B, western blots performed as described in A but with IKK2 /, RelA /, or IκBα / MEFs. 4
5 Supplementary Figure 5 A B H2AX DAPI PATM Control ATM PH2AX Dox H2AX Pac activates ATM but does not induce phosphorylated histone H2AX, an indicator of double strand DNA breaks. A, western blots of nuclear extract proteins from ZR751 cells treated with 15 μmol/l for the indicated times and probed with antibodies against phosphorylated (P ATM) or total ATM, phosphorylated (PH2AX) or total H2AX, and, as indicated. B, fluorescent confocal micrographs of ZR751 cells that were not treated (Control), treated with 5 μmol/l doxorubicin (Dox) for 4 h, treated with 15 μmol/l for 4 h, or treated with 200 nmol/l paclitaxel (Pac) for 6 h and then processed for immunofluorescence with anti H2AX antibodies or stained with the DNAbinding molecule 4,6diamidino2phenylindole (DAPI). 5
6 Supplementary Figure 6 A ATRDN B DNAPK Ub p53 Ub p53 ATR / ATRDN DNAPK / DNAPK / does not require the PI3 kinaselike kinases ATR or DNAPK to promote accumulation of ubiquitinated. A, western blots of nuclear extract proteins from cells possessing wild type ATR (ATR / ) or a dominantnegative mutant ATR protein (ATRDN) and treated with 15 μmol/l for 3 h. B, western blots as in A except that the cells either possessed (DNAPK / ) or lacked (DNAPK / ) wild type DNAPK protein. 6
7 Supplementary Figure 7 Viability inhibition (%) HCT116 HCT116 p53 / Dox Cis Sensitivity of HCT116 colon cancer cells to the cytotoxic anticancer agents doxorubicin and cisplatin. HCT116 cells possessing or lacking (p53) wild type p53 protein were treated with 5 μmol/l doxorubicin (Dox) or cisplatin (Cis) for 48 h and cell viability determined spectrophotometrically using a WST1 tetrazolium dye assay. Inhibition of cell viability was determined after normalization against control (DMSOtreated) cells and plotted as the mean of triplicate experiments. 7
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SUPPLEMENTAL FIGURE LEGENDS
SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1: Endogenous interaction between RNF2 and H2AX: Whole cell extracts from 293T were subjected to immunoprecipitation with anti-rnf2 or anti-γ-h2ax antibodies
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Supplementary Discussion The cell cycle machinery and the DNA damage response network are highly interconnected and co-regulated in assuring faithful duplication and partition of genetic materials into
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Supplementary Figure S1 Supplementary Figure S2
Supplementary Figure S A) The blots shown in Figure B were qualified by using Gel-Pro analyzer software (Rockville, MD, USA). The ratio of LC3II/LC3I to actin was then calculated. The data are represented
Supplementary Figure 1
Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna 40 20 0 % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1
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Received 28 September 2010/Returned for modification 19 October 2010/Accepted 27 April 2011
MOLECULAR AND CELLULAR BIOLOGY, July 2011, p. 2774 2786 Vol. 31, No. 14 0270-7306/11/$12.00 doi:10.1128/mcb.01139-10 Copyright 2011, American Society for Microbiology. All Rights Reserved. A Cytosolic
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