File S1 - Subverting ER-Stress towards Apoptosis by Nelfinavir and Curcumin Coexposure Augments Docetaxel Efficacy in Castration Resistant Prostate Cancer Cells

Table S1, IC50 values of individuals drugs on C4-2B and PC-3 cell survival. Figure S1, (A). Cytotoxic effects of drug combination in PC-3 cells. Percent change in cell viability following 72 hr exposures to DTX (10 nM) alone or in combination with NFR (5 µM) and/or CUR (5 µM) (n = 3; ***, p<0.0005). (B) Effect of drug combination on XBP-1 mRNA in C4-2B cells. Spliced XBP-1 mRNA expression in C4-2B cells was determined by qRT-PCR analysis. Cells were exposed to drugs for 30 min or 3 hrs, total RNA isolated, reverse-transcribed and PCR amplified. Fold changes (ΔCt) in XBP1 mRNA were calculated after normalization to GAPDH mRNA levels (n = 3). Figure S2, Temporal effects of drug combinations on AKT and ER stress in C4-2B cells. Immunoblots show the effects of drug exposure for 30 min, 3 hrs and 6 hrs. Temporal effects on (A). IGF-1 induced p-AKT and t-AKT; (B) p-eIF2α and t- eIF2α; (C) BiP/Grp78; (D) CHOP; (E) ATF4 and (F) TRIB3 levels are shown. Band intensities were normalized to β-actin levels. Treatment specific changes (lanes 2–8) are expressed as compared to controls (lane-1). Figure S3, Proposed mechanism for the antitumor efficacy of triple-drug combination. Simultaneous exposure to the DTX, NFR and CUR drug combination induces severe ER-stress, resulting in the up-regulation of CHOP, ATF4 and TRIB3. The augmented TRIB3 level suppresses the AKT survival pathway and further enhances ER-stress induced apoptosis by TRIB-3 induced caspase-3 activation. Therefore, coexposure to physiological concentrations of NFR & CUR can increase the susceptibility of CRPC cells to DTX therapy. Methods S1, (1). PC-3 Cell culturing; (2). qRT-PCR analysis of XBP-1.
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