Fig. 5

Autophagy inhibition reciprocally promotes autophagy in stem cell-like tumor subpopulations and regulates senescence to maintain stemness. a CD133/PROM1^HIGH patient-derived GBM cells were treated with 10 µM of Spautin-1 and the number of cells were counted after 24 h using trypan blue exclusion. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant. b Western blot analysis of CD133/PROM1^HIGH patient-derived GBM cells following treatment with 10 µM of Spautin-1 for CASP3 (Pro-CASP 35 kDa and cleaved CASP-3 17 & 19 kDa) and cleaved PARP (89 kDa). Positive control of cells treated with cytotoxic chemical were run in parallel. Graph represents densitometry quantification of western blots from three independent experiments. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant. c Western blot analysis of CD133/PROM1^HIGH patient-derived GBM cells following treatment with 10 µM of Spautin-1 for p21/CDKN1A expression. Graph represents densitometry quantification of western blots from three independent experiments. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant. d Non-treated controls and Spautin-1 (10 µM) treated CD133/PROM1^HIGH patient-derived GBM cells were subjected to β-galactosidase/GLB1 staining 24-h post-treatment to detect the presence of senescent cells. e Non-treated controls and Spautin-1 (10 µM) treated CD133/PROM1^HIGH patient-derived GBM cells were subjected to western blot analysis for the expression of BMI1. Graph represents densitometry quantification from three independent experiments. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant. f Non-treated controls and Spautin-1 (10 µM) treated CD133/PROM1^HIGH patient-derived GBM cells were subjected to neurosphere formation analysis and the graph represents the quantification of neurospheres > 50 µm in size. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant. g Non-treated controls and Spautin-1 (10 µM) treated CD133/PROM1^HIGH patient-derived GBM cells were subjected to glucose uptake assay and the fluorescent intensity of intracellular 2-NBDG was quantified. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant. h Extracellular lactate levels were quantified in non-treated controls and Spautin-1 (10 µM) treated CD133/PROM1^HIGH patient-derived GBM cells using a lactate assay and normalized to total protein concentration. Statistical analysis was performed using two-sided students t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ns = non-significant