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1 DOI: /ncb1864 Figure S1 Apak specifically inhibits p53 transcriptional activity. Transcription activity of p53 was measured in U2OS (p53 wild-type) and H1299 (p53 deficient) cells which were transfected with or without exogenous p53 (50 ng) together with a pg13l reporter plasmid (15 ng), an internal control reporter plasmid prl-cmv (1 ng), and increasing doses of Apak or mock vector pcmv- Myc such that that total amount of material transfected was equal. Data are mean ± s.d. (n = 3). Apak overexpression inhibits the expression of the p53 target gene Bax. Lysates from U2OS and H1299 cells transfected with Apak with or without p53 were analyzed with the antibodies indicated in the figure. Apak could not affect transcriptional activity of c-jun and NFκB. The indicated luciferase reporter plasmids (from Stratagene) and Apak were co-transfected into U2OS cells. Luciferase activity was measured as in (a). Overexpression of Apak reduces the expression of the subset of p53 target genes related to apoptosis. ChIP assays in Apak-overexpressed or control p53 +/+ HCT116 cells. 1

2 Figure S2 Apak interacts with p53. Reciprocal co-immunoprecipitation of exogenous Apak with exogenous p53 in HEK293 cells. The indicated whole cell lysate (WCL) and immunoprecipitates were analyzed by western blot using anti-flag antibody (top) to identify p53 and anti-myc antibody (bottom) to identify Apak. Subcellular localization of endogenous Apak in MCF-7 cells. Immunofluorescence analysis with anti-apak rabbit polyclonal antibody was performed. Nuclei were stained with DAPI. Images were captured by confocal microscope and merged. Scale bar, 10 μm. Distribution of endogenous Apak in MCF7 cells was determined by cell fractionation. The fractions were subjected to western blot with the indicated antibodies. Fibrillarin is a nucleolar marker protein. Co-localization of Apak and p53 in the nucleus of MCF7 cells. GFP-tagged Apak and RFP-tagged p53 were cotransfected into MCF7 cells. Images were captured by confocal microscope at 36 h post transfection. Scale bar, 10 μm. Interaction site mapping on Apak revealed by GST pull-down assays. The assays were performed similarly as described in Fig. 2a. 2

3 Figure S3 Apak attenuates p53 acetylation whereas has little effect on p53 stability. Apak overexpression attenuated p53 acetylation (K382-Ac) and TSA treatment could inhibit Apak function. Apak had little effect on the protein level of p53. Apak was either overexpressed (left panels) or downregulated by RNAi (right panels) in U2OS cells, and p53 protein levels were analyzed by western blot. Apak and HDM2 were either single or double knocked down in p53 +/+ HCT116 cells. mrna levels of p53 target genes were determined by quantitative real-time PCR. Apak and HDM2 themselves were also analyzed to show the efficiency and specificity of knockdown. Treatment of Nutlin-3, the specific inhibitor of p53-hdm2 interaction, could not significantly affect the regulation of Apak on apoptosis. Apoptosis was determined by Annexin-V staining. 3

4 Figure S4 Apak regulates p53 dependent of ATM. The repression activity of Apak on p53 in ATM wild type and mutated cells. p53 activity was measured 36 h post transfection through reporter gene assays in mock-transfected cells and in cells transfected with Apak. Ectopic expression of Apak and p53 were detected by western blot with the indicated antibodies. Apak truncate N1 (containing KRAB domain alone) could not display dominant-negative role on full-length Apak. Reporter gene assays (left) and Co-IP assays (right) were performed to determine whether the co-expression of Apak-N1 could had significant effects on the repression activity of full-length Apak on p53 and ATM-Apak interaction, respectively. Left (lanes 1-4), Anti-phospho-Apak antibodies (Ab-1, Ab-2) specifically recognize the Ser68-phosphorylated Apak. U2OS cells were transfected with Myc-Apak WT or S68A mutant and the whole cell lysates were analyzed by western blot with the two individual antibodies, Ab-1 and Ab-2, which were obtained against the antigens Ag-1 and Ag-2, respectively. Right (lanes 5-8), phosphorylated peptides of Apak (53-71 and 41-71, respectively, shown at the bottom) could block the signal of correlated polyclone antibodies. Due to the higher specificity of antibody Ab-1, it was used in Fig.5j, 5k, 6a and S4d. To further confirm the specificity of anti-apak-pser68 antibody and investigate physiological Apak phosphorylation, endogenous Apak proteins were immunoprecipitated with Apak antibody in cell lysates prepared from the MMS treated or control HCT116 (p53 +/+ ) cells. Both Apak antibody and phospho-apak antibodies were used in the western blot analysis. The results showed that although total Apak proteins were easily detected in unstressed cells, Apak phosphorylation could not be detectable, suggesting the specificity of the phospho-antibody. 4

5 Figure S5 Full scans of Western blot data. 5

6 Figure S5 continued 6

7 Supplementary Information Methods Plasmid constructs The restriction endonucleases used in the subcloning are listed below. Detailed information of the constructs and primer sequences are available upon request. (i) pcmv-myc-apak (including wild-type and various N-terminal and C-terminal deletion mutants, including N1(1-70), N2(1-245), N3(1-513), C1( ), C2( ), C3(72-688), and S16A, S68A, S68D point mutants) were digested with Bgl II and Kpn I; pegfp-c3-apak (GFP-Apak) was digested with Sal I and Bam HI; (ii) pdsred1-n1-p53 (RFP-p53) was digested with Eco RI and Bam HI. pgex-4t-2-p53 (GST-p53) was digested with Xho I and Bam HI. pflag-cmv-2-p53 was digested with Eco RI and Bam HI. (iii) pet28(a)-apak (including wild-type and two deletion mutants, N1(1-70) and C3 ( )) were digested with Sal I and Bam HI. Chromatin immunoprecipitation (ChIP) assay ChIP was performed as described 1 with some modifications. A total of p53 +/+ HCT116 cells (48 h after transfected) were crosslinked with 1% formaldehyde for 10 min at 37, followed by quenching with glycine (final concentration M) for 5 min. Cells were harvested in cell lysis buffer (5 mm PIPES ph 8.0, 85 mm KCl, 0.5% NP-40, protease inhibitors). After centrifugation, the cell pellet was resuspended in 1 ml 1

8 of Nuclei lysis buffer (50 mm Tris-Cl ph 8.1, 10 mm EDTA, 1% SDS, protease inhibitors), followed by sonication to an average DNA length of 500 1,000 bp. The sample was cleared by centrifugation as whole cell extract with 200 µl being saved for input control. After precleared with 80 µl protein-a beads for 1 h at 4, p53 antibodie (5 µl) were added to each of the sample tubes, which were then rotated at 4 C overnight. 60 µl protein-a beads was then added to each of the tubes, which then were further rotated for 1 h at 4 C. The beads were washed once with low salt wash buffer ( 50 mm HEPES ph 7.5, 140 mm NaCl, 1 % Triton X100, 0.1 % Deoxycholate sodium, protease inhibitors), once with high salt wash buffer (50 mm HEPES ph 7.5, 500 mm NaCl, 1 % Triton X100, 0.1 % Deoxycholate sodium, protease inhibitors), once with LiCl wash buffer (10 mm Tris ph 8.0, 250 mm LiCl, 0.5 % NP-40, 0.5 % Deoxycholate sodium, 1 mm EDTA) and twice with 1 ml of TE buffer. The beads were eluted with 300 µl of elution buffer (1% SDS_0.1 M NaHCO 3 ) and incubated for 5 h at 65 C to reverse crosslinks. The elutes were treated with proteinase K and extracted with phenol_chloroform followed by ethanol precipitation. The DNAs were dissolved in TE buffer and analyzed by PCR. Input DNA was diluted 1:10 for PCR. The PCR products were amplified for cycles. Primer sequences used for all ChIPs have been published 2-7. Supplementary References 1. Tanaka, T., Ohkubo, S., Tatsuno, I. & Prives, C. hcas/cse1l associates with chromatin and regulates expression of select p53 target genes. Cell 130, (2007). 2

9 2. Huang, J. et al. Repression of p53 activity by Smyd2-mediated methylation. Nature 444, (2006). 3. Hermeking, H. et al sigma is a p53-regulated inhibitor of G2/M progression. Mol. Cell 1, 3-11 (1997). 4. Kastan, M.B. et al. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, (1992). 5. Shi, X. et al. Modulation of p53 function by SET8-mediated methylation at lysine 382. Mol. Cell 27, (2007). 6. Kaeser, M.D. & Iggo, R.D. Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo. Proc. Natl. Acad. Sci. U S A. 99, (2002). 7. Oda, E. et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, (2000). 3

10 Table S1. List of potential p53 interacting proteins Bait_ Bait-Ac Prey_ Prey-Ac Prey-description In- Hits Known symbol symbol frame interaction TP53-Δ TAD TP53-Δ TAD TP53-Δ TAD TP53-Δ TAD NM_ UBE2I NM_ Ubiquitin-conjugating enzyme E2I (UBC9 homolog) NM_ APOH NM_ Apolipoprotein H (β-2-glycoprotein I) NM_ BMP1 NM_ Bone morphogenetic protein 1 NM_ PIASx NM_ Protein inhibitor of activated STAT, x Y 2 Y Y 1 N Y 1 N Y 1 Y TP53-Δ NM_ Apak/ NM_ KRAB type zinc finger Y 1 N TAD ZNF420 protein 420 This table shows the interaction results of the yeast two hybrid screening. The 1 st and 2 nd column is the gene symbol and the Genbank accession number of the bait, respectively. The 3 rd and 4 th column is the gene symbol and Genbank accession number of the prey genes, respectively. The 5 th column is the description of the preys. The 6 th column is the information of prey gene that is in reading frame or out of frame. The 7 th column is the number that multiple prey sequences represent the same gene and the last column means whether the interactions have been previously reported or novel ones. 4