Supporting Information Su et al. 10.1073/pnas.1211604110 SI Materials and Methods Cell Culture and Plasmids. Tera-1 and Tera-2 cells (ATCC: HTB- 105/106) were maintained in McCoy s 5A medium with 15% FBS (Gibco). All other cell lines used in this study were cultured in DMEM plus 10% FBS. Rnf2 cdna was cloned into prk5-flag vector or pgex- 4T1 vector. p53 cdna was cloned into prk5-myc vector or pet-28a vector. HA Mdm2 construct was provided by Dr. Shengcai Lin. Rnf2ΔRing cdna (amino acid 91 336) was cloned into prk5 FLAG vector. Bmi1 and Bmi1Δ1 44 cdna was cloned into pet-28a vector. The Rnf2 point mutants were generated using a site-directed mutagenesis kit (Transgen). Antibodies and Reagents. Anti-p53 (ab17990) and anti-rnf2 (ab3832 and ab28629) antibodies were purchased from Abcam. Anti-p53 (DO-1) antibody was fromsantacruz.anti-mdm2 (OP-46) was from Calbiochem. Anti-Bmi1 (05-637) was from Millipore. Anti-FLAG, anti-ha, anti-lamin, anti-tubulin, and anti-actin antibodies were from Sigma. Doxorubicin (D1515), CHX (C4859), and puromycin (P8833) were from Sigma. MG132 was from Calbiochem. Primers for Q-PCR. p21: forward-agcgaccttcctcatcca, reverse-agcctctactgccaccat; p53: forward-gactg- ACATTCTCCACTTCT, reverse-tctgacgcacacctattg; Rnf2: forward-gtgtgtggatgtgtgttt, reverse-atgtg- GCTATGTATATGTTACC; BAX: forward-tcagatgtggt- CTATAATG, reverse-caatgaacttgagcaatt; Mdm2: forward-gtcttctcttaggtcacat, reverse-acaggcaatt- ACAATCTTAC; GAPDH: forward-tcctggtatgacaacg- AAT, reverse-tcttcctcttgtgctctt; and Puma: forward- TGTGAATCCTGTGCTCTG, reverse-cccaaatgaatgcca- GTG. RNA Interference. Rnf2 control shrna, Rnf2 shrnas, and p53 shrna fragments were cloned into the psuperretro vector. The following shrna sequences were used: Control shrna sequence, TCGGTACTCAACCGTTAAG; Rnf2 shrna-1, ACGGAACT- CAACCATTAAG; Rnf2 shrna-2, TGGATGGTGCTAGTGA- AAT; p53 shrna, GACTCCAGTGGTAATCTAC; and Bmi1 shrna, GTTCACAAGACCAGACCAC. The shrna plasmids were transfected into cells as described in the text. Coimmunoprecipitation Assay. The cells were lysed in lysis buffer (20 mm Tris HCl, ph 7.5, 100 mm NaCl, 1 mm EDTA, 1% Nonidet P-40) containing fresh protease inhibitors. Whole cell lysates were incubated with 2 μg of antibody and protein A or protein G Sepharose beads (Millipore) for 2 h at 4 C. The immunocomplexes were subsequently washed with lysis buffer four times and separated through SDS/PAGE. GST Precipitation Assay. GST or His fusion proteins were prepared following standard protocol. GST Rnf2 fusion proteins bound to the GSH Sepharose were incubated with purified His-p53. After washing for five times, the bound proteins were separated by SDS/PAGE. In Vitro Binding Assay. In vitro translated fusion proteins were prepared following standard protocol (Promega). FLAG Rnf2 fusion proteins bound to the anti-flag M2 affinity gel were incubated with 35 S-labeled p53 fragments. After washing for five times, the bound proteins were separated by SDS/PAGE. Immunofluorescence. Tera-1 cells were plated on glass coverslips and transfected with indicated constructs. After 72 h with puromycin treatment, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with indicated antibodies. In Vivo Ubiquitination Assay. HCT116 cells were transfected with the indicated plasmids. The cells were treated with MG132 for 6 h, harvested, and lysed with RIPA buffer supplemented with 10 mm iodoacetamide (GE Healthcare) and protease inhibitors. Endogenous p53 was immunoprecipitated with 1 μg of anti-p53 polyclonal antibodies and immunoblotted with anti-ha or antip53 (DO-1) antibodies. In Vitro Ubiquitination Assay. A 100 μl sample of the in vitro translated FLAG-tagged Rnf2 (TNT, Promega) was purified using FLAG beads and incubated with bacteria expressed Bmi1 for 1 h. After extensive washing, the reconstituted complex was used for in vitro ubiquitination without elution from the FLAG beads. We incubated 2 μl in vitro translated 35 S-labeled p53 (TNT, Promega) in a 30 μl reaction mixture containing 50 mm Tris HCl (ph 7.5), 5 mm MgCl 2, 0.6 mm DTT, 2 mm ATP, 100 ng of ubiquitin-activating enzyme E1, 200 ng of ubiquitin-conjugating enzyme UbcH5c, 10 μg of ubiquitin (Calbiochem), and 2 μg of the Rnf2 Bmi1 complex. After incubation at 37 C for 2 h, the reactions were boiled and separated on an 8% SDS/PAGE gel. The 35 S-labeled p53 was detected using autoradiography. Cytosal/Nuclear Fractionation. Cells were transfected with indicated constructs. At 48 or 72 h after transfection, cells were harvested and washed with PBS one time. Cytoplasmic proteins were extracted by lysing with solution A (10 mm Hepes, ph 7.9, 10 mm KCl, 1.5 mm MgCl 2, 0.34 M sucrose, 10% glycerol, 1 mm DTT, 0.1% Triton X-100) for 5 min. The intact nuclei were then lysed in RIPA buffer. Cell Cycle Analysis. Cells transfected with indicated shrnas were trypsinized at day 4, fixed with 70% ethanol for at least 2 h at 4 C. Cells were then washed with PBS, resuspended in 300 μl of PBS containing DNase-free RNase (100 μg/ml), and incubated at 37 C for 30 min. Propidium iodide (10 μg/ml, Sigma) was used for DNA staining. Samples were analyzed by flow cytometry (BD Pharmingen) and cell cycle analyses were performed by ModFit software. Apoptosis Assays. Cells transfected with indicated shrnas were fixed with 4% paraformaldehyde for 1 h and measured by TUNEL staining using an in situ cell death detection kit, TMR (Roche Applied Science). Apoptotic cell numbers were analyzed by flow cytometry using APC-conjugated annexin V (BD Pharmingen) staining. Colonogenic survival assay was performed by seeding 1 10 6 cells transfected with indicated shrnas in culture medium supplemented with 1 μg/ml puromycin. After 3 wk, colonies were fixed with 4% paraformaldehyde and stained with 0.2% methylene blue for visualization. Tissue Microarray. The tissue arrays of human ovarian cancer samples were purchased from US Biomax (BC11115). Immunohistochemical staining of Rnf2, p53, and Mdm2 were scored by pathologists in a blinded manner. 1of6
Fig. S1. Rnf2 negatively regulates p53 related to Fig. 1. (A) Rnf2 interacts with the DNA binding domain of p53. Indicated p53 DNA plasmids were used to translate their respective proteins in a TNT-coupled Reticulocyte Lysate system. The translated 35 S p53 polypeptides were incubated with FLAG or FLAG-Rnf2 M2 Affinity Gel. Proteins retained on the gels were resolved in SDS/PAGE and revealed by autoradiogram. (B) Relative mrna levels of Rnf2 in transfected Tera- 1 cells. (C) Rnf2 overexpression represses p53 protein levels in Tera-1 cells. Tera-1 cells transfected with indicated plasmids were treated with Doxorubicin for 2 h before harvest. Cell lysates were subjected to SDS/PAGE and blotted with indicated antibodies. (D) Rnf2 protein sequences are highly conserved among human, mouse, and zebrafish. (E) p53 up-regulation in Rnf2 depleted cells can be reversed by zebrafish Rnf2 or Mdm2 cotransfection. Tera-1 cells were transfected with indicated constructs. Cells were lysed and cell lysates were blotted with indicated antibodies 72 h later. 2of6
Fig. S2. Rnf2 regulates p53 in specific cell types related to Fig. 2. (A) Rnf2 depletion increases p53 protein levels in Tera-2, MCF-7 cells, and MEFs but not in HeLa or PA-1 cells. Tera-2, MCF-7, HeLa, or PA-1 cells transfected with control or Rnf2 shrna constructs were harvested 3 d after puromycin treatment. Rnf2 flox/flox MEFs infected with control or CMV-Cre expression adenovirus were harvested 2 d after infection. Cell lysates were then subjected to immunoblotting. (B) Rnf2 interacts with p53 in HEK 293 cells. HEK 293 cell lysates were subject to immunoprecipitation with control IgG or anti-p53 antibodies. The immunoprecipitates were then blotted with indicated antibodies. (C) Expression levels of Rnf2 and p53 in different cell lines. (D) Rnf2 is not a direct target of p53. Tera-1 cells treated with doxorubicin were harvested at the indicated time. Proteins were extracted and subjected to Western blot. Fig. S3. Rnf2 poly-ubiquitinates p53 in cell nucleus related to Fig. 3. (A and B) Rnf2 promotes nuclear p53 poly-ubiquitination in vivo. HCT116 cells transfected with indicated constructs were treated with MG132 for 4 h before harvest. The cells were harvested and fractionated, cytoplasmic, or nuclear p53 was immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with an anti-ha monoclonal antibody. (C) Bmi1 is required for Rnf2-mediated p53 ubiquitination. HCT116 cells transfected with indicated constructs were treated with MG132 for 4 h before harvest. p53 was immunoprecipitated with antip53 polyclonal antibodies and immunoblotted with monoclonal anti-ha or anti-p53 antibodies. 3of6
Fig. S4. Rnf2 regulates p53-mediated cell growth arrest and apoptosis in specific cells related to Fig. 4. (A and B) Rnf2 regulates cell growth through p53 pathway. (A) Tera-1 cells transfected with indicated constructs were fixed and stained as described. (B) Quantification of p-h3 positive cells in A. Error bars represent the SEM of triplicate experiments. **P < 0.01; *P < 0.05 two-tailed Student t test. (C and D) Rnf2 depletion does not cause G1 arrest and apoptosis in 293 andhelacells. HeLa or 293 cells transfected with indicated constructswere treated with puromycin for 3 d. Cells were then harvested andcell-cycle profiles or apoptosis were determined by FACS. 4of6
Fig. S5. Rnf2 regulates ovarian carcinoma cell growth through p53 related to Fig. 5. (A and B) Rnf2 regulates ovarian cancer cell growth. A2780 cells transfected with indicated constructs were plated, and cell numbers were then counted at the indicated time. (C) Cell cycle analysis of A2780 cells stably expressed with indicated shrnas. (D and E) Immunostaining of p-h3 was performed with A2780 cells transfected with the indicated shrna constructs. (E) Quantification of p-h3 positive cells in D. Error bars represent the SEM of triplicate experiments. **P < 0.01; *P < 0.05 two-tailed Student t test. (F) Rnf2 ubiquitinates p53 mutants. HCT116 cells (p53 negative) transfected with indicated constructs were treated with MG132 for 4 h before harvest. p53 mutants were immunoprecipitated with anti-myc polyclonal antibodies and immunoblotted with monoclonal anti-ha or anti-p53 antibodies. (G) Rnf2 interacts with p53 mutants. HCT116 cells (p53 negative) transfected with indicated constructs were lysed and subject to immunoprecipitation with antiflag monoclonal antibody. The immunoprecipitates were then blotted with the indicated polyclonal antibodies. 5of6
Table S1. Statistic analysis of Rnf2 expression in normal and ovarian carcinoma tissues Tissue types No. tissue samples Rnf2 positive (%) Rnf2 negative (%) Normal ovarian tissue 10 0 (0%) 10 (100%) Ovarian serous cystoadenoma 72 63 (87.5%) 9 (12.5%) Ovarian mucinous carcinoma 10 9 (90%) 1 (10%) Ovarian clear cell carcinoma 5 3 (60%) 2 (40%) Rnf2 expression is up-regulated in ovarian carcinoma cells-related to Fig. 5. The table shows quantifications of Rnf2, p53, and Mdm2 expression levels in ovarian carcinoma samples. Table S2. Statistic analysis of p53 expression in normal and ovarian carcinoma tissues Tissue types No. tissue samples p53 positive (%) p53 negative (%) Normal ovarian tissue 10 Ovarian serous cystoadenoma 72 27 (37.5%) 44 (61.1%) Ovarian mucinous carcinoma 10 1 (10%) 9 (90%) Rnf2 expression is up-regulated in ovarian carcinoma cells-related to Fig. 5. The table shows quantifications of Rnf2, p53, and Mdm2 expression levels in ovarian carcinoma samples. Table S3. Statistic analysis of Mdm2 expression in normal and ovarian carcinoma tissues Tissue types No. tissue samples Mdm2 normal (%) Mdm2 up-regulation (%) Normal ovarian tissue 10 10 (100%) 0 (0%) Ovarian serous cystoadenoma 72 48 (66.7%) 24 (33.3%) Ovarian mucinous carcinoma 10 10 (100%) 0 (0%) Ovarian clear cell carcinoma 5 5 (100%) 0 (0%) Rnf2 expression is up-regulated in ovarian carcinoma cells-related to Fig. 5. The table shows quantifications of Rnf2, p53, and Mdm2 expression levels in ovarian carcinoma samples. 6of6