FIGURE 5.

UPR signaling, proteasomal impairment, and oxidative stress in human A375 malignant melanoma cells exposed to aurin. A and C, aurin-modulation (10 μm; ≤24 h) of DDIT3 mRNA levels (panel A), total and phospho-PERK (Thr-980), total and phospho-eIF2-α (Ser-51), CHOP (panel B), and total protein ubiquitination (panel C) were assessed by immunoblot analysis. In panel B exposure to thapsigargin (T; 300 nm; 6 h) served as a positive control. Immunoblot analysis representative of three independent repeats is depicted. D, luminescence-based analysis of proteasomal chymotrypsin-like enzymatic activity in A375 cells exposed to aurin ((1–10 μm, 3- and 6-h time points). E, analysis of oxidative stress in aurin-exposed A375 cells (10 μm, ≤24 h) as monitored by flow cytometric detection of 2′,7′-dichlorodihydrofluorescein (DCF) fluorescence. One set of histograms representative of three repeats is shown. F, time course of aurin modulation of intracellular reduced glutathione content in A375 cells (10 μm, ≤18 h) normalized to cell number (mean ± S.D., n ≥ 3). G, after preincubation (24 h) with l-buthionine-S,R-sulfoximine (BSO; 1 mm), aurin-induced (5 μm, 24 h) A375 cell death was assessed by flow cytometric analysis of AV/PI-stained cells (mean ± S.D., n ≥ 3).