Fig. 2: Cytotoxic effect of doranidazole on GSCs.

a Flow cytometric analysis of cell death for GSC-H cells exposed to 0 or 3 mM doranidazole under normoxic or hypoxic conditions for 3 days. Representative profiles and quantification of PI-positive cells are shown. FSC forward scatter. b Flow cytometric analysis of cell death for GSC-H cells exposed to 3 mM metronidazole (Metro), 3 mM misonidazole (Miso), or dimethyl sulfoxide (DMSO) vehicle under normoxic or hypoxic conditions for 3 days. c Immunofluorescence staining for EF5 (marker of hypoxic cells) in a sphere formed by GSC-H cells. Nuclear staining, Hoechst 33342. d GSC-H spheres formed over 7 days were exposed to the indicated concentrations of doranidazole for 12 or 24 h, after which dead cells were identified based on PI uptake. Representative images and quantification of the PI-positive sphere area at 24 h are shown. e Evaluation of cell death based on PI uptake for GSC-H spheres incubated for 24 h with the indicated drugs or DMSO vehicle. Quantification of the PI-positive sphere area is shown. f Evaluation of cell death based on PI uptake for GSC-H spheres incubated for 24 h with the indicated drugs or DMSO vehicle. Quantification of the PI-positive sphere area is shown. g Immunofluorescence staining for EF5 incorporation in spheres formed by U251 and Becker cells over 2 days. Nuclear staining, Hoechst 33342. h Evaluation of cell death based on PI uptake for U251 and Becker spheres incubated for 24 h with the indicated concentrations of doranidazole. Representative images and quantification of the PI-positive sphere area are shown. Quantitative data for (a, b) are means ± s.d. for n = 3 independent experiments and were analyzed with the paired two-tailed Student’s t-test (a) or one-way ANOVA followed by Dunnett’s post hoc test b. Data for d–f, h are means ± s.d. of six biologically distinct replicates in a representative experiment (n = 3 independent experiments performed) and were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05, ***P < 0.001. Scale bars, 100 µm (c, g) 300 µm (d, h).