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HSPB8 Supplemental Data Cdk-independent role of cyclin D1 in radiation sensitivity Trent and Schmidt - Supplemental information page 1 HSPB8 Supplemental Data Supplemental Materials and Methods Coimmunoprecipitation experiments: Westerns for p53, actin and p14ARF were performed as described in methods. Antibodies used included anti-p53 (DO1; Santa Cruz, Santa Cruz, CA), anti-p14ARF (14P02; Abcam, Cambridge, MA), and the control actin antibody as described in methods. Purified positive control p14ARF protein was the generous gift of Dr. James Rocco (MGH Cancer Center; Boston, MA). For coimmunopreciptiations, cells transfected with the vector control, or the cyclin D1 or KE mutation expression plasmids were lysed in ELB lysis buffer, centrifuged and immounoprecipitated with 100 μg of anti-cyclin D1 preconjugated to protein A sepharose beads. The beads were washed five times and bound proteins were eluted by boiling in SDS-sample buffer and resolved by 12% SDS-PAGE. The immunoprecipitated proteins were then detected in a Western analysis using antibodies to CDK4 (rabbit polyclonal C-22 Santa Cruz, Santa Cruz, CA), p21 (Rabbit polyclonal C-19 Santa Cruz, Santa Cruz, CA) or cyclin D1 (Rabbit polyclonal M-20, Santa Cruz, Santa Cruz, CA). Review of array analyses: Pre-calculated meta-analyses evaluated linkage of HSPB8 to estrogen receptor positive tumors were identified using software available at www.Oncomine.org (1-12). The pre-calculated box plots describing the normalized expression units for HSPB8 were downloaded for display. RNA Expression Analysis The RNase protection assay was done using an RNase protection assay III kit (Ambion, Austin, TX) and labeled RNA probes generated with hAPO3d template sets (BD PharMingen, San Diego, CA) according to the manufacturer's instructions. Two micrograms of mRNA harvested from the indicated MCF7 transfectants before and 48 hours after 4 Gy of radiation were analyzed in this assay. Cdk-independent role of cyclin D1 in radiation sensitivity Trent and Schmidt - Supplemental information page 2 qrtPCR assays for candidate genes thought to be co-regulated by cyclin D1 and CEBPβ were performed using TaqMan Low Density ARrays (TLDA); Applied Biosystems). Genes and their corresponding assay numbers included the following: CCND1-Hs00277031_m1, DNAJA3 Hs001170600_m1, HSPA1A Hs00271244_s1; HSPA8 Hs00852842_gH, HSPH1 Hs00971475_m1, STIP1 Hs00428979_m1, and PGR Hs00172183_m1. Samples were prepared according to the manufacturer’s instructions and run using the Applied Biosystems 7900HT system. Supplemental Figure Legends: Supplemental Figure 1. Cyclin D1 overexpression has no effect on p53 levels after irradiation. MCF7 cells expressing the indicated constructs (3.1, D1 and KE) were exposed to 0, 2, 6 and 20 Grey radiation. The cells were then harvested 18 hours after irradiation and protein lysates were prepared for Western blots. A single autoradiographic exposure was obtained for each of the first thee panels of data (A-C) in this figure. The individual autoradiographs were then cut and re- aligned to show changes in protein levels across doses for each construct. 3.1 designates the vector control cells. D1 designates the cyclin D1 transfectants. KE designates the KE mutant cyclin D1 transfectants. (A) Western analysis for p53 levels is shown. (B) The same blot is re-probed for actin as a loading control. (C) The same blot is probed with anti-p14ARF except that 10 ng of control p14ARF protein lysate is included in the last lane as a positive control (TP). (D) The KE mutant of cyclin D1 was originally described as incapable of interacting with Cdk4/6 (13), which we confirmed in immunoprecipitations of the stable cyclin D1 transfectants that were probed for Cdk 4 (K4 – second row). We then assessed the ability of the KE mutant to interact with p21 in the absence of interactions with Cdk 4 and found that substantial amounts of p21 were found in a Western analysis in association with the KE mutant of cyclin D1 (second row – p21). The Cdk-independent role of cyclin D1 in radiation sensitivity Trent and Schmidt - Supplemental information page 3 amounts of cyclin D1 present in the immunoprecipitation for cyclin D1 were similarly assessed (row one – D1). Supplemental Figure 2: HSPB8 is highly associated with estrogen receptor positive breast cancers. Thirteen microarray analyses were evaluated using software developed at Oncomine (14). Shown are the box plots for all studies. Statistical significance of studies shown are as follows: 1. 6.2 x 10-7, (1); 2. 2.5 x 10-6 (2); 3. 2.8 x 10-6 (3); 4. 5.1 x 10-5 (4); 5. 3.2 x 10-4 (5); 6. 0.003 (6); 7. 0.003 (7); 8. 0.013 (8); 9. 0.017 (9); 10. 0.028 (10); 11. 0.034 (11); 12. 0.065 (12); and 13. 0.096 (6). Supplemental Figure 3: Cyclin D1 does not regulate death receptor 5 or CEBPβ-dependent target gene expression in transfected MCF 7 cells. (A) An RNAse protection assay evaluated expression of the death receptor pathway mRNAs in MCF7 transfectants. mRNA isolated from MCF 7 cells transfected with vector (V), cyclin D1 (D), KE cyclin D1 mutant KE (K) or the amino terminal truncated cyclin D1 (C) was analyzed for effects on death receptor pathway mRNAs. Identified are the protected bands corresponding to Fas-ligand (Fas-L), DcR-1 and death receptors 3, 4, and 5 (DR3, DR4, and DR5). The undigested probes that correspond to the identified bands are identified by a connecting line. Lanes 1-4 used mRNA harvested before radiation treatment and lanes 7-10 contained mRNAs isolated after radiation. (B) The mRNA response of five genes previously identified as CEBPβ-dependent targets of cyclin D1 regulation were evaluated using quantitative real time PCR (qrtPCR) and compared with HSPB8, which was identified as a cyclin D1 target in invasive breast cancers. Fold changes are shown for MCF 7 cells treated with estradiol, cells transfected with cyclin D1 and cells treated with fulvestrant. The graphs identify the mean and standard error for three determinations in each case, and the mean number is shown for each determination. HSPB8 was previously shown by us to be Cdk-independent role of cyclin D1 in radiation sensitivity Trent and Schmidt - Supplemental information page 4 estradiol and cyclin D1-responsive, and the data are re-plotted here for comparison with the other genes under evaluation (30). Five candidate cyclin D1 and CEBPβ-regulated genes include DNAJA3, HSPA1A, HSPA8, HSPH1, and STIP1. Positive controls include cyclin D1 (CcnD1) and the progesterone receptor (PGR). Supplemental References: 1. van de Vijver MJ, He YD, van't Veer LJ, et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 2002;347(25):1999- 2009. 2. Hess KR, Anderson K, Symmans WF, et al. Pharmacogenomic predictor of sensitivity to preoperative chemotherapy with paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide in breast cancer. J Clin Oncol 2006;24(26):4236-44. 3. Wang Y, Klijn JG, Zhang Y, et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 2005;365(9460):671-9. 4. Miller LD, Smeds J, George J, et al. An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival. Proc Natl Acad Sci U S A 2005;102(38):13550-5. 5. Sotiriou C, Neo SY, McShane LM, et al. Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci U S A 2003;100(18):10393-8. 6. Zhao H, Langerod A, Ji Y, et al. Different gene expression patterns in invasive lobular and ductal carcinomas of the breast. Mol Biol Cell 2004;15(6):2523-36. Cdk-independent role of cyclin D1 in radiation sensitivity Trent and Schmidt - Supplemental information page 5 7. Sotiriou C, Wirapati P, Loi S, et al. Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 2006;98(4):262-72. 8. Perou CM, Jeffrey SS, van de Rijn M, et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A 1999;96(16):9212-7. 9. Yu K, Ganesan K, Miller LD, Tan P. A modular analysis of breast cancer reveals a novel low-grade molecular signature in estrogen receptor-positive tumors. Clin Cancer Res 2006;12(11 Pt 1):3288-96. 10. Richardson AL, Wang ZC, De Nicolo A, et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 2006;9(2):121-32. 11. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A 2003;100(14):8418-23. 12. Pollack JR, Sorlie T, Perou CM, et al. Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors. Proc Natl Acad Sci U S A 2002;99(20):12963-8. 13. Hinds PW, Dowdy SF, Eaton EN, Arnold A, Weinberg RA. Function of a human cyclin gene as an oncogene. Proc Natl Acad Sci U S A 1994;91(2):709- 13. 14. Rhodes DR, Kalyana-Sundaram S, Mahavisno V, et al. Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia (New York, NY 2007;9(2):166-80. .
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