Ks (eight doses) of treatment applying the IVIS imaging system. The mice had been euthanized 48 hours immediately after the final injection, and primary tumors have been excised and weighed. A portion of the tumors was in liquid nitrogen for molecular HSP90 Antagonist supplier evaluation and yet another portion was formalin fixed and paraffin embedded. In any instance, please clarify how liquid nitrogen was used for immunohistochemistry for routine hematoxylin and eosin staining and TUNEL assay as described previously.36 The remaining tumor tissue was stored at -80 until use. Statistical evaluation. The data were expressed as the means SD of 3 or extra independent experiments, and statistical evaluation was performed applying the two-tailed and paired Student’s t test. P 0.05 was CB2 Modulator supplier considered statistically substantial and indicated by an asterisk. Supplementary material Figure S1. Dose-dependent downregulation of Bcl-2 protein in MDA-MB231 tumors soon after single NL-Bcl-2 siRNA injection (iv. tail vein). Figure S2. Therapeutic silencing of Bcl-2 by only 3 i.v. injections of NL-Bcl-2 siRNA inhibits in vivo tumor growth of ER(-) MDA-MB-231 xenografts in nude mice (p0.05). Figure S3. Therapy schedules with siRNA and chemotherapy in mice bearing tumors. Figure S4. A) Dose-dependent inhibition of MDA-MB-231 cells by doxorubicin (72h). B) Doxorubicin induces autophagy in MDA-MB-231 cells as indicated by acridine orange staining and FACS evaluation (48h). C) Doxorubicin induces apoptosis and autophagy in MDA-MB-231 cells as indicated by Annexin V/PI and acridine orange staining and FACS evaluation (48h). D) Knockdown of autophagy genes like ATG5 and Beclin 1 inhibits doxorubicin-induced autophagy in MDA-MB-231 cells. Acknowledgments. This operate was funded by a Susan Komen Breast Cancer Award (BO) and, in aspect, by the NIH (grants U54 CA096300, U54 CA151668, P50 CA083639, the DOD (grant BC085265) and An NCI institutional Core Grant (CA16672).1. 2. three. 4. 5. six. 7. Youle, RJ and Strasser, A (2008). The BCL-2 protein household: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9: 479. Yip, KW and Reed, JC (2008). Bcl-2 family proteins and cancer. Oncogene 27: 6398406. Korsmeyer, SJ (1999). BCL-2 gene household plus the regulation of programmed cell death. Cancer Res 59(7 Suppl): 1693s700s. Buchholz, TA, Davis, DW, McConkey, DJ, Symmans, WF, Valero, V, Jhingran, A et al. (2003). Chemotherapy-induced apoptosis and Bcl-2 levels correlate with breast cancer response to chemotherapy. Cancer J 9: 331. Patel, MP, Masood, A, Patel, PS and Chanan-Khan, AA (2009). Targeting the Bcl-2. Curr Opin Oncol 21: 51623. Shimizu, S, Kanaseki, T, Mizushima, N, Mizuta, T, Arakawa-Kobayashi, S, Thompson, CB et al. (2004). Role of Bcl-2 family proteins within a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol six: 1221228. Tawfik, K, Kimler, BF, Davis, MK, Fan, F and Tawfik, O (2012). Prognostic significance of Bcl-2 in invasive mammary carcinomas: a comparative clinicopathologic study among “triple-negative” and non-“triple-negative” tumors. Hum Pathol 43: 230.8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.Tabuchi, Y, Matsuoka, J, Gunduz, M, Imada, T, Ono, R, Ito, M et al. (2009). Resistance to paclitaxel therapy is related with Bcl-2 expression via an estrogen receptor mediated pathway in breast cancer. Int J Oncol 34: 31319. Tanabe, K, Kim, R, Inoue, H, Emi, M, Uchida, Y and Toge, T (2003). Antisense Bcl-2 and HER-2 olig.