Fig. 3: DOXY-mediated inhibition of mitochondrial protein synthesis impairs cell viability and reduces oncogenic transcription factors across a panel of neuroblastoma cells.

a, b Expression of mitochondrial proteins, mitochondrial-encoded MT-CO1 and nuclear-encoded proteins ATP5A1 and TOMM20, after 24-h DOXY treatment analyzed by western blotting (a) and subsequent densitometry (b). Normalized protein levels are plotted relative to untreated controls, mean ± SD. c, d MTT cell viability assay analysis of 6-day treatment with DOXY and other FDA-approved antibiotics targeting procaryotic ribosomes, tigecycline, chloramphenicol, and linezolid (c), and targeting bacterial cell wall synthesis, ampicillin (d). Calculated IC₅₀ are indicated. Data presented as mean ± SD, biological n = 4, technical n = 3. e MTT and f CellTiter-Glo cell viability assay analysis after 72-h treatment revealed all neuroblastoma cell lines to be highly sensitive to DOXY. Respective IC₅₀ values are indicated in brackets. Data points are mean ± SD, biological n ≥ 3, technical n = 3. g Cell death rate of neuroblastoma cells (CHLA-15 and CHLA-20) and neonatal dermal fibroblasts (NDF-2 and NDF-3) was analyzed after 72 h of DOXY treatment by flow cytometry using SYTOX Red staining. Data are presented as the difference in percentages of SYTOX Red-positive dead cells after indicated treatment vs. respective untreated controls, biological n = 3. h, i Densitometric analysis (h) of western blotting detection (i) revealed downregulation of c-MYC, N-MYC, and HIF-1α across a panel of neuroblastoma cell lines treated with indicated concentrations of DOXY for 24 h. Normalized protein levels are plotted relative to untreated controls, mean ± SD. Densitometric analysis of HIF-1α is provided in Fig. S4. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test (b, g, h), *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001.