Fig. 8.

a Anti-UBC9, anti-PIAS1, and anti-SUMO2/3 antibody immunoblotting of total cellular lysates from control (siNON), UBE2I- (siUBE2I), or PIAS1-silenced (siPIAS1) VCaP cells in C (37 °C) or HS (30 min at 43 °C) conditions. b Effect of UBC9 or PIAS1 depletion on the gene expression of select HS-inducible (HSPA1A, HSPA1B, HSPA6, JUN, FOS) and HS-repressed (CTDSP1, ATG2A) target genes. Quantitation of mRNA was done using RT-qPCR with target- specific primers. Measurements were normalized to GAPDH mRNA levels, and fold changes were calculated in reference to siNON at control conditions. Error bars are standard deviations of three independent biological replicates. Stars depict statistical significances of pair-wise comparisons of each time point (purple, siUBE2I vs. siNON; green, siPIAS1 vs. siNON; *P <0.01, ***P <0.001; ANOVA and Bonferroni). c Cell survival after HS. siUBE2I-, siPIAS1-, and siNON-transfected cells were exposed to HS (43 °C) for 4 h and allowed to recover at 37 °C for 16 h, and the proportions of living cells in the samples were measured and normalized to non-HS (HS: 0). Three biological replicates are shown (black = siNON, purple = siUBE2I, green = siPIAS1) and the bars represent the mean values of each group. Stars depict statistical significances of indicated pair-wise comparisons (*P <0.05, **P <0.01; ANOVA and Bonferroni)