FIGURE 3 from Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors

Simvastatin induces p53-independent PUMA upregulation. A, A BCL2 family model figure depicting protein interactions and how BH3 mimetics can cooperate with apoptotic sensitizers. B, A representative Western blot analysis of statin-insensitive (red) and statin-sensitive (black) MMCLs treated for 16 hours with 10 µmol/L simvastatin. n = 3. C, Quantification by densitometry of PUMA upregulation. Mean ± SD, n = 3, significance (α<0.05) determined by one-sample t test of log-transformed fold changes against the value of 0. *, P < 0.05; **, P < 0.01. D, Representative (n = 3) Western blots showing that PUMA upregulation by 10 µmol/L simvastatin is not dependent on the transcriptional activity of p53. Expression of the p53 dominant negative, GSE56, was accomplished in three MMCLs, L363, NCI-H929, and OPM2 wherein the basal p53 status is mutated [*L363 cells have a point mutation in exon7/intron 7 splicing junction (base C782G) that impairs splicing of intron 7], wild-type, and mutated (R175H), respectively. GSE56 expression was confirmed with a rodent-specific p53 antibody (Cell Signaling Technology# 32532). Activation of p53 using 10 µmol/L of the topoisomerase inhibitor, etoposide, was used as a positive control to confirm blockade of p53 activation of PUMA upregulation.