FIGURE 3.

Early heat shock response gene expression followed by apoptotic cell death in human A375 malignant melanoma cells exposed to aurin. A, aurin-modulation (1–20 μm, 6 h) of HSPA1A, HSPA6, and HMOX1 mRNA levels as determined by independent real time RT-PCR analysis. B and C, modulation of heat shock protein levels in aurin-exposed A375 cells as assessed by immunoblot analysis (panel B, 10 μm, 1–24 h; panel C, 1–40 μm, 24 h). D, up-regulation of HSF transcriptional activity was assessed using an HSE-luciferase reporter construct in A375 cells (dual luciferase system; F, firefly luminescence; R, renilla luminescence; A and ATA, 10 μm; 8 h exposure time; positive control: celastrol (Cel: 500 nm). E, HSF1 nuclear translocation in response to aurin (10 μm; ≤120 min exposure time) detected by immunoblotting; N, nuclear extract; C, cytosolic extract. F and G, ATA (≤20 μm; 24 h) modulation of heat shock-related gene expression at the mRNA (quantitative RT-PCR (panel F)) and protein (Hsp70) level (panel G); for comparison, aurin-exposed (10 μm, 24 h) cells were analyzed. Immunoblot analysis representative of three independent repeats is depicted. Bar graphs represent data obtained from three independent repeats (mean ± S.D.; n ≥ 3). N.S., not significant. H, cell viability in response to aurin exposure (10 μm, 24 h) performed in the absence of presence of the pancaspase inhibitor zVADfmk (40 μm) was monitored using flow cytometric analysis (annexin V-PI staining; numbers in quadrants indicate viable (AV-negative, PI-negative) in percent of total gated cells (mean ± S.D., n = 3)).