Aminoglycoside Stress Together with the 12S rRNA 1494C>T Mutation Leads to Mitophagy
Figure 5
Mitochondrial morphology and mitophagy.
(A) Mitochondrial morphology in human 143B.TK− cells (I-1, without gentamycin stimulation; I-2, with gentamycin stimulation), the control A3t cybrid (II-1, without gentamycin stimulation; II-2, with gentamycin stimulation) and the mutant cybrid (III-1, without gentamycin stimulation; III-2 & III-3, with gentamycin stimulation). The mitochondria were visualized using TMRM fluorescence. The two small panels in the middle column show an enlarged view of the boxed regions. The arrowheads indicate fragmented mitochondria. Scale bar: 5 µm. Two independent experiments were conducted. (B) Relative expression rates of genes involved in mitochondrial dynamics in mutant cybrids without (white) vs. with (black) gentamycin stimulation. 143B.TK− was used as a normalization control (grey). (C) Western blot analysis of whole-cell extracts to detect LC3-B in 143B.TK− cells, three control cybrids (A3t, A6t and A7t) and three mutant cybrids (1494T1/T2/T3), without (−) or with (+) gentamycin stimulation. Tubulin was probed as a loading control. (D) Quantification of the protein levels (LC3-B/Tubulin) shown in panel C. (E) Relative expression rates of ATG16L1, a marker for the mitophagy process, in the control (white) and mutant cybrids (black) without (−) or with (+) gentamycin stimulation. Data was normalized to the value for 143B.TK− (grey). (F) Analysis of apoptosis in control cybrids (A3t, A6t and A7t) and mutant cybrids (1494T1/T2/T3), without (red) or with (blue) gentamycin stimulation, performed via flow cytometry.