FIGURE 7.

Aurin up-regulates early PMAIP1 gene expression causing Noxa-dependent A375 melanoma cell apoptosis. Viability of aurin-exposed cells was monitored using flow cytometric analysis (annexin V-PI staining; mean ± S.D., n = 3). A, dose-response relationship (aurin 1–20 μm, 24 h). B, time course (aurin 10 μm, 1–24 h). C, viability of A375 cells exposed to aurin (10 μm, 24 h) combined with selected caspase inhibitors and N-acetyl-l-cysteine (NAC, 10 mm, 24 h preincubation). D, aurin-induced (10 μm, 6–24 h) caspase-3 activation in A375 cells as examined by flow cytometric detection using an Alexa 488-conjugated monoclonal antibody against cleaved procaspase-3. One experiment (histogram) representative of three similar repeats is displayed. E, mitochondrial transmembrane potential was determined using flow cytometric analysis of JC-1 stained cells (time course; 10 μm, ≤24 h). Numbers indicate cells with polarized mitochondria (circle) in percent of total cells (mean ± S.D., n = 3)). F, time course of PMAIP1 expression changes induced by aurin (10 μm) at the mRNA level (quantitative RT-PCR; n = 3). G, immunoblot analysis of Noxa protein levels in aurin (10 μm; ≤24 h) exposed cells with siControl or siPMAIP1 cotreatment. H, viability of aurin-exposed (10 μm; 24 h) cells was monitored as a function of siControl or siPMAIP1 cotreatment using flow cytometric analysis (annexin V-PI staining; mean ± S.D., n = 3).