Fig. 5
Effect of combining cyst(e)inase and auranofin on oxidative stress, DNA damage markers and mitophagy. a Oxidative stress markers, and apoptosis and autophagy signaling in MIA-PaCa2 cells treated as follows: 50 nM Cys, Aur: 1 or 2 μM, Cys + Aur combination, combination + 0.5 or 1 mM GSH ethyl ester (GSH). Only the cleaved (cl.) forms of PARP, caspase (casp.) 3 and caspase 7 are shown. b The doubly tagged mCherry-GFP-LC3 protein emits both red (mCherry) and green (GFP) fluorescence (yellow when merged) from autophagosomes but only red fluorescence after fusion with lysosome due to quenching of GFP in the acidic environment of the autolysosome (top left). Confocal microscopy images of mCherry-GFP-LC3-transfected MIA-PaCa2 cells 6 h after indicated treatments (Cys: 250 nM, Aur: 0.5 μM, GSH: 2 mM; data from a representative experiment). Scale bars, 5 μm. c Confocal microscopy images of mCherry-GFP-LC3-transfected MIA-PaCa2 cells 6 h after indicated treatments (Cys: 250 nM, Aur: 0.5 μM, Bafilomycin A1: 10 nM; data are from a representative experiment). Scale bars, 5 μm. d Mitochondrial mass was analyzed by labeling MIA-PaCa2 cells with MitoTracker Green. e Quantification of MitoTracker Green fluorescence after indicated combinatorial treatments for 24 h (n = 3–4 independent experiments). f Mitochondrial membrane potential (Δψm) was analyzed by labeling MIA-PaCa2 cells with MitoTracker Green and MitoTracker Red. g Quantification of cells with defective mitochondrial membrane potential (Δψm) after indicated combinatorial treatments for 24 h (n = 4 independent experiments). All data represent mean ± s.e.m. For (e, g), Cys: 50 nM for MIA-PaCa2 and Panc1, 100 nM for BxPC3; Aur: 0.5 μM for MIA-PaCa2 and Panc1, 2 μM for BxPC3; ####P or ****P < 0.0001; compared to untreated controls (*, comparison done for Cys, Aur and Cys + Aur) or to Cys + Aur (#, comparison done for only Cys + Aur + GSH); two-way ANOVA with Bonferroni’s method for multiple-comparison test
