Fig. 2: Therapy-naive and drug-resistant neuroblastoma cells retain sensitivity to inhibitors of mitochondrial quality control.

a An overview of utilized mitochondrial inhibitors with indicated mechanism of action. b MTT cell viability assay analysis after 72-h treatment showed no significant difference in sensitivity to mitochondrial inhibitors between therapy-naive CHLA-15 and post-therapy CHLA-20 cell lines (calculated IC₅₀ values are indicated). Data are presented as mean ± SD, biological n ≥ 3, technical n = 3. c Live-cell imaging growth rate analysis of CHLA-15 and CHLA-20 treated with indicated concentrations of mdivi-1 and DOXY. Data are presented as mean ± SD, biological n ≥ 3, technical n = 3. Supporting Supplementary Videos 1, 2 are provided. d Western blotting of the cleaved caspase-3 showed that DOXY and mdivi-1 treatment for 24 h induced apoptosis in both CHLA-15 and CHLA-20. Blots are representative of three experiments. e, f Flow cytometry analysis of cell viability after DOXY treatment for 24 h. Representative histograms including percentages of SYTOX Red-positive dead cells as mean ± SD, biological n ≥ 7 (e) and the difference in percentages of SYTOX Red-positive dead cells after indicated treatment vs. untreated controls (f). g Pretreating neuroblastoma cells with 50 µM DOXY for 24 h reduced their neurosphere formation capacity. Notably, the inhibition of sphere-formation capacity was more pronounced in CHLA-20 cells which exhibit enhanced stem-like traits (3-fold reduction relative to untreated control) compared with CHLA-15 (1.6-fold reduction); biological n = 3, technical n = 3. h CellTiter-Glo cell viability assay analysis of therapy-naive SK-N-BE(1) and post-therapy SK-N-BE(2)C after 72-h treatment (calculated IC₅₀ are indicated) showed their similar sensitivity to DOXY whereas post-therapy SK-N-BE(2)C were found resistant to conventional chemotherapy drugs (see also Fig. S2) or a BH3 mimetic, ABT-737, inhibiting multiple anti-apoptotic BCL-2 proteins. Data are presented as mean ± SD, biological n = 3, technical n = 3. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test (f) and by unpaired two-tailed Student’s t-test (g), *p < 0.05, **p < 0.01, #p < 0.0001.