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. 2014 Nov 20;5(11):e1536. doi: 10.1038/cddis.2014.500

Figure 3.

Figure 3

Combination simvastatin and metformin treatment does not induce apoptosis in C4-2B metastatic CRPC cells. (a) Western blot analysis of total and cleaved caspase-3 and cleaved PARP protein expression from total cell lysates of C4-2B3 and C4-2B4 cells following treatment with 4 μM simvastatin (SIM) and/or 2 mM metformin (MET) for 24−72 h. Total cell lysates of C4-2B3 and C4-2B4 cells treated with 2 μM (S)-(+)-camptothecin (CAMP) for 48 h used as apoptosis positive control. GAPDH used as loading control. (b) C4-2B3 and C4-2B4 cells treated with 4 μM SIM and/or 2 mM MET for 48−96 h followed by staining with FITC-conjugated Annexin V (AV) and propidium iodide (PI) and analyzed by flow cytometry. Percentage of PI(−)AV(−), PI(−)AV(+), PI(+)AV(−), and PI(+)AV(+) cells demonstrated in bar graphs of mean±S.D. from triplicate samples. Representative density plots of controls and samples shown in Supplementary Figure S1. (c) Percentage cell viability (mean±S.D.) by methylene blue assay of C4-2B3 and C4-2B4 cells following treatment with combination 4 μM SIM and 2 mM MET±10 μM z-VAD-fmk pan-caspase inhibitor for 24−96 h, n=3 per treatment group. NS denotes not significant difference in cell viability between the treatment groups of SIM+MET in presence or absence of z-VAD-fmk as determined by the two-tailed Student's t-test