Fig. 4

Expression of antioxidant proteins upon cyst(e)inase treatment and their concurrent inhibition. a Comparison of proteins related to antioxidant function in the three PDAC cell lines. Nrf2 nuclear factor (erythroid-derived 2)-like 2, GCL-C glutamate-cysteine ligase catalytic subunit, xCT cystine/glutamate antiporter, TXNRD1 and TXNRD2, thioredoxin reductase 1 and 2, respectively. b, c Expression of antioxidant proteins following cyst(e)inase treatment. TBHP treatment (200 μM, 2 h) is included as a positive control for ROS accumulation b. d−f Isobologram of the effect of the combination of cyst(e)inase (Cys) and sulfasalazine d, cyst(e)inase and BSO e, and cyst(e)inase and auranofin f (data from more than three independent experiments). g Relative mitochondrial ROS (mROS) levels (red) and cell death (black) in MIA-PaCa2 cells 24 h after treatment with 50 nM Cys, 0.5 μM (mROS) or 2 μM (cell death) auranofin (Aur), their combination (Cys + Aur), and the combination plus 0.5 mM GSH ethyl ester (GSH) (for mROS, n = 5 independent experiments; for cell death, n = 3 independent experiments). h Relative mitochondrial ROS (mROS) levels (red) and cell death (black) in BxPC3 cells 24 h after treatment with 100 nM Cys, 2 μM (mROS) or 4 μM (cell death) Aur, their combination (Cys + Aur), and the combination plus 0.5 mM GSH ethyl ester (GSH) (for mROS, n = 5 independent experiments; for cell death, n = 3 independent experiments). i Regulatory cell cycle proteins 24 h after the indicated combinatorial treatments in MIA-PaCa2 and BxPC3 cells. For (b, c), “+” represents 250 nM cyst(e)inase treatment except for (c), where “+” for Panc1–48 h represents 100 nM treatment. ####P or ****P < 0.0001; for (g, h), compared to untreated controls (*, comparison done for Cys, Aur and Cys + Aur) or to Cys + Aur (#, comparison done for only Cys + Aur + GSH); one-way ANOVA with Bonferroni’s method for multiple-comparison test. Experiments in (a, b, c, i) were performed twice. All other experiments were repeated three times or more