Fig. 7: Overexpression and phosphorylation of MYC proteins correlate with sensitivity to cell death via mitochondrial ISR, conserved across various models of nervous system tumors.

a MTT cell viability assay analysis was used to determine sensitivity of cell lines derived from various nervous system tumors to DOXY. Respective IC₅₀ values for 72-h treatment are indicated in brackets. Data presented as mean ± SD, biological n ≥ 3, technical n = 3. b–d Densitometric analysis (b) of immunoblots (c, d) from lysates after 24-h DOXY treatment showed dose-dependent induction of mitochondrial stress and ISR in a panel of cell lines derived from different nervous system tumors. Contrary to neuroblastoma, this did not lead to consistent downregulation of c-MYC, although HIF-1α was significantly downregulated in nervous system tumors. Normalized protein levels are plotted relative to untreated controls, mean ± SD; N.D. – not detectable. Complete densitometric analysis is provided in Fig. S8. e Western blotting detection and densitometric analysis of c-MYC, p-c-MYC(T58), and p-c-MYC(S62) revealed that CHLA-20 neuroblastoma cells have significantly higher c-MYC level and extensively enhanced phosphorylation of c-MYC compared with Daoy, NSTS-5 and NDF-3 cells that were less sensitive to DOXY. Normalized protein levels are plotted relative to untreated control, mean ± SD. f Western blotting detection of c-MYC, p-CMYC/N-MYC(T58) and p-c-MYC(S62) in a panel of nervous system tumors. g A significant positive correlation between the percentage of dead cells after 72-h treatment with 25 μM DOXY and the basal levels of p-CMYC/N-MYC(T58) in untreated neuroblastoma models and Daoy medulloblastoma cells, r = Pearson correlation coefficient; ǂ, MYCN-amplified. Related flow cytometry and protein densitometry data are provided in Fig. S10. h Western blotting detection of N-MYC and p-c-MYC/N-MYC(T58) (upper panel; densitometric analysis is provided in Fig. S12a) and cell death rate flow cytometric analysis (lower panel) revealed that Tet21N MYCN-off cells with extensively downregulated N-MYC and p-c-MYC/N-MYC(T58) levels are significantly less sensitive to DOXY-induced cell death compared with N-MYC overexpressing Tet21N MYCN-on cells. Cell death rate was analyzed using SYTOX Red staining after 24-h treatment with 50 µM DOXY. Data are presented as percentages of SYTOX Red-positive dead cells after indicated treatment, biological n = 5. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test (b, e, h) and by Pearson correlation (g), *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001.