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. Author manuscript; available in PMC: 2017 May 21.
Published in final edited form as: Nat Med. 2016 Nov 21;23(1):120–127. doi: 10.1038/nm.4232

Figure 2. Effect of Cyst(e)inase in PCa cells.

Figure 2

(a–c) HMVP2 cells were treated with indicated concentrations of Cyst(e)inase. (a) Cell viability as assessed by alamar blue assay at indicated time points. (b) Relative cellular ROS levels 4 h post-treatment as assessed by 2′,7′-Dichlorofluorescin diacetate (DCFDA) fluorescence; (c) Relative GSH levels 24 h post-treatment as assessed by spectrophotometric analysis. (d–i) HMVP2 cells were treated with indicated concentrations of Cyst(e)inase for 24 hours. (d,e,f,h,i) Metabolic stress markers and regulatory cell cycle proteins were measured by immunoblot. Immunoblots were performed at least three times with β-actin controls for each experiment. Numbers above blots indicate band intensities relative to untreated conditions. Images have been cropped for presentation, uncropped images are shown in Supplementary Fig. 14. (g) Cell-cycle phase distribution was analyzed by Guava-based flow cytometry 24 h post treatment. (j) DU145 PCa cells were treated with indicated concentrations of Cyst(e)inase and/or N-Acetyl-L-Cysteine (NAC) for 48 h and cell viability was assessed by crystal violet assay. For (a–c, g, j): data are expressed as mean ± s.e.m; *P<0.05; ***P<0.001; ****P<0.0001; one-way ANOVA followed by Tukey’s multiple comparison test (a–c, g) or Bonferroni’s multiple comparison test (j).