Fig. 1.

Effect of cyst(e)inase treatment on survival of PDAC cell lines. a Relative cell survival 48 h after cyst(e)inase treatment in three pancreatic cancer cell lines (n = 3 cultures for each dose). b Intracellular l-Cys levels upon treatment with 250 nM cyst(e)inase (one treated and one untreated culture for each time point, data from a representative experiment). c Intracellular GSH levels 24 h after treatment (n = 3 cultures for each dose). d Cellular ROS levels as assessed by 2′,7′-dichlorofluorescin diacetate (DCFDA) fluorescence 24 h after treatment. tert-Butyl hydrogen peroxide (TBHP) treatment is included as a positive control for ROS accumulation (n = 3–5 independent experiments). e Mitochondrial ROS levels as assessed by MitoSOX fluorescence following 24 h cyst(e)inase treatment (n = 3–6 independent experiments). f Oxidative stress signaling via JNK and ATM, and apoptosis signaling following cyst(e)inase treatment. Cys cyst(e)inase. A white line is added to separate the TBHP treatment group with the rest of the time course data. Original blots can be found in the Supplementary Figures. g Cell death in Panc1 cells as assessed by trypan blue staining 48 h after treatment (n = 3 independent experiments). h Cell death in MIA-PaCa2 and BxPC3 cells 72 h after cyst(e)inase treatment (n = 3–4 independent experiments). i Cellular growth assay of Panc1, MIA-PaCa2, and BxPC3 cells (n = 3 cultures for each time point) with cyst(e)inase treatment. For f, “+” represents 250 nM cyst(e)inase treatment. All data represent mean ± s.e.m. except for b. Dotted line in a represents the 95% confidence band for the fitted curve (four-parameter logistic model). *P < 0.05, ****P < 0.0001; compared to untreated controls (c–e, g, h), or between the bottom plateaus of the fitted curve (a); two-way ANOVA (c–e) or one-way ANOVA (a, g, h) with Bonferroni’s method for multiple-comparison test. Experiments in f were repeated twice with similar results. All other experiments were repeated three times or more