A flag-hausp expression vector was kindly provided by Dr. Wei Gu (Columbia

Transcription

1 Supplementary Materials and Methods Plasmids A flag-hausp expression vector was kindly provided by Dr. Wei Gu (Columbia University). GST-HAUSP (1-212) was generated by PCR amplification of the first 636 bp at the 5 of HAUSP sequences and inserted into the pgex 4T vector. The cdna was synthesized by RT-PCR from MCF-7 cell mrna and then inserted into the plenti6 vector or the psg5 expression vector. Lentiviral cdna library construction: A MCF-7 cdna library was constructed using Gateway Technology according to the manufacturer s instruction (Invitrogen). The entire entry cdna library was then shuttled into a plenti6 destination vector to generate a lentivirus expression library. Lenti6 vectors were then transfected into 293FT cells to produce lentivirus particles as described previously (Wu et al., 212). Screening of SRC-3 downstream apoptosis-resistant genes MCF-7shSRC-3 cells were infected with the lentiviruses containing the cdna library for two days and then were grown in DMEM medium under the selection of antibiotics (7.5 g/ml blasticidin) and.5 mm SNP. Genomic DNA was then isolated from cell clones that survived using a Qiagen DNA blood kit. The inserted sequence was PCR amplified by primers flanking the insertion site in the plenti6 vector and sequenced. The mrna levels of the identified genes in sisrc-3 and scramble sirna (Dharmacon) treated MCF-7 were measured by real time quantitative RT-PCR to assess the effects of SRC-3 on the transcription of these genes. A stable MCF-7shSRC-3 cell line harboring flag tagged was established by the infection of lentivirus and selection with 7.5 g/ml blasticidin.

2 SNP sensitivity 5 cells were seeded in a 96-well plate and a series of SNP concentrations were added in triplicate to the cells the next day. The percentage of surviving cells was assessed one day later using a MTS assay according to the Manufacturer s instruction (Promega). GST pull down E.coli expressed GST-HAUSP or GST was bound to 1 l glutathione sepharose 4B followed by the addition of deletion mutant expression vector-transfected 293T cell extracts. The procedure is essentially the same as described previously (Yi et al., 25). In vitro deubiquitination 293T cells were transfected with expression vectors for flag-p53, HA-Ubiquitin and MDM2. 24 hours after transfection, cells were treated with 2 M of proteasome inhibitor MG132 for four hours before being lysed. The ubiquitinated flag-p53 was purified using flag M2 beads. After extensive washing, the flag antibody bead-bound p53 was then incubated with purified HAUSP from baculovirus (produced by Dan L. Duncan Cancer Center Proteomics Core facility) in the absence or presence of protein in a deubiquitination buffer (5 mm Tris- HCl ph 8., 5 mm NaCl, 1 mm EDTA, 1 mm DTT, 5% glycerol) for 1 h at 37 C. HAUSP was pre-incubated with on ice for 1 min before the reaction. After the reaction, the flag beads were washed with a wash buffer, boiled, and then loaded to a SDS-PAGE gel. The levels of p53 ubiquitination were detected by Western blot using a HA-specific antibody. Breast cancer cdna array analysis

3 The breast cancer cdna array (Origene) contains 48 samples covering normal and different stages of breast carcinoma. The expression levels of SRC-3 and in these samples were analyzed by quantitative PCR and were normalized against the levels of -actin. The primers and Taqman probes for SRC-3 and used in the qpcr were obtained from Applied Biosystems gene assays. The mean value of SRC-3 or levels in these samples was set as 1 or its log 2 value is. The scatter plot of SRC-3 and expressions, the correlation coefficient and the p-value were analyzed using statistics software from Wessa ( Quantitation Gel bands and Western blots were normalized against s ( -actin), and quantification was performed using Scion Image. p value was calculated by student t test.

4 Yi_Fig S1 SRC-3 -actin shgfp shsrc-3 Fig. S1 Depletion of SRC 3 in MCF 7 cells increased cell sensitivity to doxorubicin treatment. Left panel, the level of SRC 3 protein in shsrc 3 stable cells compared to the (shgfp) cells as determined by the Western blot analysis. Right panel, percentage of surviving cells under different concentration of doxorubicin treatment.

5 Yi_Table S1 Gene Gene description ALDOA aldolase A, fructose-bisphosphate CASKIN2 CASK interacting protein 2 CAPZB capping protein (actin filament) muscle Z-line, beta CFL1 cofilin 1 (non-muscle) CTSD cathepsin D (lysosomal aspartyl peptidase) DDX54 DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 EEF1A1 eukaryotic translation elongation factor 1 alpha 1 FLJ11151 hypothetical protein FLJ11151 KRT18 keratin 18 LLGL2 lethal giant larvae homolog 2 (Drosophila) PRKRIP1 PRKR interacting protein 1 (IL11 inducible) PSMB4 proteasome (prosome, macropain) subunit, beta type, 4 PTBP1 polypyrimidine tract binding protein 1 RBM4 RNA binding motif protein 4 RPS5 ribosomal protein S5 RTEL1 regulator of telomere elongation helicase 1 SMC6 structural maintenance of chromosomes 6 TNF receptor-associated factor 4 Table S1: Genes identified from the lentiviral cdna library rescue screening.

6 A SRC-3 -actin shluc shsrc-3 Relative mrna level SRC 3 shsrc 3 Relative mrna level shluc shsrc3 Yi_Fig S2 B SNP sisrc C GFP SRC-3 SRC-3 SRC actin actin Fig. S2 SRC 3 regulated expression (A) transcription was decreased in SRC 3 shrna stably transfected MCF 7 cells. Left panel: Western blot analysis of SRC 3 protein levels in or shsrc 3 stable MCF 7 cells. Right panel: the mrna levels of SRC 3 and as assessed by real time qpcr. (B) SRC 3 knockdown decreased protein levels. MCF 7 cells were treated with scramble or sisrc 3 (2 nm) for three days and then treated with 2mM SNP for four hours and cell lysates were subjected to Western blot analysis. (C) SRC 3 overexpression by adenovirus infection significantly increased protein level. A549 cells were infected by GFP or SRC 3 expressing adenovirus for three days before harvesting.

7 A SRC 3 B SRC 3 Yi_Fig S3 Low High Fig. S3: Two examples of SRC 3 and expressions in breast tumor array. Upper panel, grad II III invasive ductal carcinoma, low SRC 3 and low expressions. Bottom panel, grade III invasive ductal carcinoma, high SRC 3 and high expressions. (A) lower magnification (B) higher magnification

8 A Luciferase activity kb promoter.4 kb promoter B Relative mrna level c fos sifos Relative mrna level sifos Yi_Fig S4 C sirna sifos sirna Primers: 1 kb promoter Primers: 1 kb promoter Primers: 3 UTR input c fos SRC 3 input c fos SRC 3 input c fos SRC 3 Fig. S4: SRC 3 directly regulates transcription through coactivating the AP1 transcription factor. (A) SRC 3 enhanced the transcriptional activity of AP 1 (c jun/c fos) on upstream 1 kb promoter driven luciferase but not the upstream.4 kb promoter driven luciferase reporter (B) Knockdown of c fos decreased mrna levels in MCF 7 cells (C) ChIP assay for the recruitment of c fos and SRC 3 to the 1 kb promoter but not the 3 UTR region. Knockdown of c fos significantly decreased the recruitment of both c fos and SRC 3

9 Yi_Fig S5 A OD49nm Mouse mammary tumor cells sirna si si B Percent of cell survival ZR75 1 cells sirna si SNP concentration (mm) SNP concentration (mm) C percentage of cell survival LNCaP cells SNP concentration (mm) D flag Percent of cell survival MCF 7 cells doxorubicin concentration ( M) flag E percentage of cell survival LNCaP cells flag Doxorubicin concentration ( M) Fig. S5 expression levels were important for regulating cell sensitivities to cytotoxic stresses (A) knockdown by sirna sensitized mouse mammary tumor cells to SNP induced death. (B) Knockdown of sensitized ZR75 1 cells to SNPinduced death (C) stably overexpressing LNCaP cells were more resistant to SNP treatment. (D E) stably overexpressing MCF 7 shsrc 3 cells (D) and LNCaP (E) cells were more resistant to doxorubicin induced death compared to cells. represents p<.5.

10 A Annexin V PI B Yi_Fig S6 No treatment SNP Annexin V PI positive positive No treatment 6±2 1±1 SNP 116±9 3±2 Doxorubicin 229±26 2±5 Doxorubicin Fig. S6 SNP treatment induced MCF 7 cell apoptosis. Live cells treated with 4 mm of SNP or 2 Mof doxorubicin for 4 hours and were then subjected to Annexin V or Propidium iodide (PI) staining. Normal viable cells are not stained with either Annexin V or PI. Annexin V positive and PI negative cells are early apoptotic cells. Both Annexin V and PI positive cells are necrotic or late apoptotic cells. (A) fluorescent images of live MCF 7 cells (B) Quantification of Annexin V or PI positive cells per field

11 Yi_Fig S7 A vector SRC-3 B si C si #1 #2 SRC SNP p p53 p53 actin actin actin D E p53 p actin actin Fig. S7 (A) SRC 3 overexpression in A549 cells significantly decreased the p53 protein level in the presence of 4 mm SNP. A549 cells were transiently transfected with a SRC 3 expressing plasmid or an empty vector as a. Cells were treated with 4 mm SNP for 4 hours before harvesting. (B) knockdown significantly increased p53 induction by SNP in U2OS cells. (C) p53 protein levels were significantly increased by two different stealth si (Invitrogen) upon SNP treatment in MCF 7 cells. (D E) p53 protein level was decreased in overexpressing LNCaP stable cells upon 4mM SNP (D) or 2 M doxorubicin (E) treatment.

12 A MCF 7 cells B 12 MCF 7 cells Yi_Fig S sisrc 3 sisrc 3+sip si si+sip53 C doxorubicin concentration LNCaP cells D SNP concentration (mm) LNCaP cells SNP concentration (mm) sip53 flag Doxorubicin concentration ( M) sip53 flag Fig. S8: p53 is important for SRC 3 and mediated cell resistance to cytotoxic agents. (A) Knockdown of p53 increased SRC 3 depleted MCF 7 cell resistance to doxorubicin treatment. (B) Knockdown of p53 increased depleted MCF 7 cell resistance to SNP treatment. (C D) p53 depleted or overexpressed LNCaP cells have similar responses to SNP (D) or doxorubicin (E) treatment

13 Yi_Fig S9 A Relative mrna level Relative mrna level p53 -SNP +SNP si si B IgG C 1/1 input IP: flag IP: IgG HAUSP HA + + Myc MDM2 Flag HAUSP + Fig. S9: (A) knockdown did not alter p53 mrna levels. Left panel: mrna levels in scramble sirna or si transfected cells in the absence or presence of SNP as assessed by real time q PCR. Right panel: p53 mrna levels in scramble sirna or si transfected cells in the absence or presence of SNP. (B) Endogenous interacted with HAUSP in LNCaP prostate cancer cells. Co IP was performed using either antibody or IgG. (C) overexpression did not alter the interaction between MDM2 and HAUSP significantly. 293T cells were transiently transfected with Myc MDM2 and flag HAUSP in the absence or presence of HA co transfection. Cell lysates were IP with anti flag antibody or IgG. MDM2 was detected using antimyc antibody.

14 Yi_Fig S1 Breast Cancer Nuclear + Normal Breast tissue Cytoplasm + X2 X4 Fig. S1 (A) had more nuclear staining in breast tumors compared to predominant cytoplasmic staining in normal breast tissues. Upper panel, lower magnification; bottom panel, higher magnification

15 Yi_Fig S1 BID Case 1 Case 2 Fig. S1 (B) Two examples of and BID expressions in ER positive breast tumors

16 Yi_Fig S11 SNP vector MDM MDM2 p actin Fig. S11 MDM2 overexpression significantly decreased p53 protein level in the absence of SNP but not in the presence of SNP. MCF 7 cells was transfected with MDM2 expression vector or empty vector and then treated with SNP for 4 hours before harvesting. p53 protein level was analyzed by Western blot using p53 specific antibody.