Fig. 4: DOXY-mediated inhibition of mitochondrial translation disrupts mitochondrial morphology and fission machinery and primes mitochondria for apoptosis.

a Mitochondrial morphology–visualized by immunofluorescence staining of TOMM20 (cyan)–revealed signs of fragmentation and swelling in most cells after 24-h treatment with 50 µM DOXY. At this timepoint, apoptosis marked by cleaved caspase-3 (yellow) and fragmented nuclei (TO-PRO-3; magenta) was detected only in a subset of cells. Maximum intensity projections of confocal microscopy Z-stacks are shown. b Image analysis by ImageJ plug-in tool MiNA - Mitochondrial Network Analysis confirmed disrupted mitochondrial morphology in cells treated with 50 µM DOXY for 24 h. Data are presented as parameters determined for individual field of vision images, mean ± SD. c Flow cytometry using JC-1 probe after 24-h DOXY treatment revealed dose-dependent loss of mitochondrial potential. Upper panel, representative contour plots; percentages of cells with depolarized mitochondria are presented as mean ± SD, biological n = 3. Bottom panel, the differences in percentages of cells with depolarized mitochondria after indicated treatment vs. untreated cells. d, e Western blotting detection (d) and densitometric analysis (e) of proteins related to mitochondrial dynamics and BCL-2 anti-apoptotic proteins after 24-h DOXY treatment in indicated concentrations. Normalized protein levels are plotted relative to untreated controls, mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test (b) and by one-way ANOVA followed by Tukey’s multiple comparisons test (e), *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001.