Withaferin a

Withaferin A (WA, purity > 95%) was purchased from ChromaDex (Irvine, CA), dissolved in dimethyl sulfoxide (DMSO; 20 mM stock), and stored at −80°C. CIS and anti-β-Actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). CIS was dissolved in normal saline. Cell culture media and fetal bovine serum (FBS) were purchased from MediaTech (Manassas, VA) and Atlanta Biologicals (Norcross, GA), respectively. Other reagents needed for cell culture were purchased from Invitrogen-Life Technologies (Carlsbad, CA). Antibodies against phospho(S428)-ATR, ATR, ATR interacting protein (ATRIP), phospho(S345)-checkpoint kinase 1 (CHK1), CHK1, and phospho(S10)-histone H3 were purchased from Cell Signaling Technology (Danvers, MA). Anti-phospho(T1989)-ATR antibody was from GeneTex (Irvine, CA). Anti-manganese superoxide dismutase (MnSOD) antibody was from EMD Chemicals (Gibbstown, NJ). Annexin V/propidium iodide (PI) assay kit for apoptosis detection was purchased from BD Biosciences (San Jose, CA).

Withaferin A (WFA) was purchased from Chromadex (ASB-00023250–250) and stored in aliquots at −20° C dissolved in dimethyl sulfoxide (DMSO). Injured mice were treated with vehicle (DMSO) or WFA (2 mg/kg solubilized in DMSO) on the day of injury, and every subsequent day, by intraperitoneal (i.p) injection for the duration of 7 or 14 days post injury [12 (link)].

Withaferin A (WA, purity > 95%) was purchased from ChromaDex (Irvine, CA) and dissolved in dimethyl sulfoxide (DMSO). Culture media were purchased from MediaTech (Manassas, VA). Fetal bovine serum and antibiotics were purchased from Invitrogen-Life Technologies (Carlsbad, CA). Annexin V/propidium iodide assay kit for apoptosis detection was purchased from BD Biosciences (San Jose, CA), whereas Cell Death Detection ELISA^PLUS kit was from Roche Diagnostics (Indianapolis, IN). Antibodies against Pin1 and Cdc25C were purchased from Cell Signaling Technology (Danvers, MA); anti-Cyclin B1 and anti-Cdc2 antibodies were from Santa Cruz Biotechnology (Dallas, TX); anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from GeneTex (Irvine, CA); and anti-β-Actin and anti-β-Tubulin antibodies were from Sigma-Aldrich (St. Louis, MO). Recombinant human Pin1 protein was purchased from MyBioSource (San Diego, CA). Human Apoptosis Antibody Array was from Abcam (Cambridge, MA).

MDA-MB-231, MDA-MB-231-pLKO.1, MDA-MB-231-DR5^shRNA1 and MDA-MB-231-DR5^shRNA2 xenografts were generated as previously described (24 (link)), grouped in 2 experimental groups (8 mice/group) and treated with intraperitoneal injections of either vehicle (10% DMSO, 40% cremophor-EL, and 50% PBS) or vehicle containing 4 mg Withaferin A (ChromaDex Inc., Irvine, CA)/kg body weight 5days/week for 5 weeks. The dose and route of WFA administration were selected from previous study documenting in vivo efficacy of WFA (8 (link)). Tumors were collected after 4 weeks of treatment; measured, weighed, and subjected to further analysis by immunohistochemistry, RT-PCR and western blotting. At least four random, nonoverlapping representative images from each tumor section from eight tumors of each group were captured using ImagePro software for quantitation of pERK, pRSK, CHOP, pElk1, and DR5 expression. MMTV-neu mice model- Mammary tumor tissues from our previously published prevention study in MMTV-neu mice (11 ) were also used to determine the expression of these proteins by western blotting. In this study, WFA administration resulted in a statistically significant decrease in macroscopic mammary tumor size, microscopic mammary tumor area (11 ). All animal studies were in accordance with the guidelines of Johns Hopkins University IACUC and University of Pittsburgh IACUC.