Fig. 3: Mechanism of doranidazole-induced GSC death.

a Evaluation of cell death based on PI uptake for GSC-H spheres incubated for 24 h with 3 mM doranidazole and either DMSO vehicle or the cell death inhibitors (100 µM) necrostatin-1 (Nec-1), ferrostatin-1 (Fer-1), or Z-VAD-FMK (Z-VAD). Representative images and quantification of the PI-positive sphere area are shown. b Evaluation of cell death as in (a) for GSC-H spheres incubated for 24 h with 3 mM doranidazole and the indicated concentrations of deferoxamine (DFO). c Evaluation of cell death as in (a) for GSC-H spheres incubated for 24 h with 3 mM doranidazole and SKQ1 (0 or 10 µM). Flow cytometric analysis of MitoSOX Red staining in GSC-H cells incubated with or without 3 mM doranidazole and 10 µM SKQ1 under normoxic (d) or hypoxic (e) conditions for 12 h. Representative profiles as well as quantification of the mean fluorescence intensity (MFI) of MitoSOX Red from n = 5 (d) or n = 3 (e) independent experiments are shown. f, g Flow cytometric analysis of BODIPY 581/591 C11 staining in GSC-H cells incubated with or without 3 mM doranidazole and 10 µM SKQ1 under normoxic (f) or hypoxic (g) conditions for 18 h. Representative profiles and quantification of the MFI of BODIPY 581/591 C11 from n = 3 independent experiments are shown. Quantitative data are means ± s.d. from six biologically distinct replicates in a representative experiment, n = 3 independent experiments performed a–c or from the indicated number of independent experiments d–g and were analyzed by one-way ANOVA followed by Dunnett’s post hoc test a, b, the unpaired two-tailed Student’s t-test (c), the paired two-tailed Student’s t-test d, f, or one-way ANOVA followed by Tukey’s post hoc test e, g. *P < 0.05, ***P < 0.001. Scale bars, 300 µm (a–c).