Inhibition of HSP27 in LN443 cells suppresses survival signaling, promotes death signaling, and decreases colony forming efficiency, eliminates SPARC-induced pro-death signaling in temozolomide (TMZ), and decreases survival in TMZ. A. Representative Western blots of LN443 cells (left panel) treated with control siRNA (Csi) or HSP27 siRNA (HSP27si) and H2 cells treated with control siRNA (Csi) and C1.1 cells treated with HSP27 siRNA (HSP27si) (right panel) for 72 hr. For both panels, equal numbers of siRNA-treated cells were plated overnight in growth serum, and then treated with 0 (0.1% DMSO control), 40, 80 or 100 μM TMZ for 2 days before lysing. Arrows indicate ≥ 2-fold increases or decreases due to HSP27 siRNA treatment. # - Indicates a ≥ 2-fold increase in the ratio of LC-3II/LC-3I. Asterisks indicate ≥ 2-fold increases or decreases due to TMZ treatment. Note: SPARC refers to endogenous SPARC in LN443 cells and SPARC-GFP in H2 cells, and 43/72 refers to the 43 kDa endogenous SPARC, whereas 72 kDa refers to SPARC-GFP. B. Means for colony forming efficiency ± SD of LN443 cells ± HSP27 siRNA plating 750 cells/60-mm dish. *** p = 0.018. C. Means for surviving fraction ± SD of LN443 cells ± HSP27 siRNA ± increasing concentrations of TMZ plating 750 cells/60-mm dish. * Csi vs. HSP27si: 20 μM; p = 0.0031, 40 μM; p = 0.0092, 60 μM; p = 0.0006, 80 μM; p = 0.0011, and 100 μM; p = 0.0053. Plating 1500 cells gave similar results.