Figure 3.

Mitochondrial dysfunction following senescence in HepG2 and Huh7 cells. (A) Representative transmission electron micrographs of control and senescent hepatoma cells. Scale bar = 1 μm. (B) Representative histogram plots showing changes in mitochondrial polarization as measured by sequestration of TMRE in Control and senescent (Sen) cells on sixth day as analyzed by flow cytometry. Bar diagrams shows quantification of TMRE in control and senescent hepatoma cells using 3 biological replicates. (C) Representative images of control and senescent cells stained with mitochondrial potential dependent dye and Mitotracker red (mitochondria, red) and counterstained with DAPI (nucleus, blue). Scale bars = 100 μm and 50 μm. (D) Bar diagram showing quantification of mitochondrial ROS production in control and senescent cells stained with fluorescent dye, MitoSOX, which is a probe to detect mitochondrial superoxide. (E) mtDNA quantification in HepG2 and Huh7 cells by real-time qPCR amplification of mitochondrial encoded 12S rRNA and the nuclear encoded 18S rRNA, which served as a reference gene. Results are expressed as relative ratio of 12S mtDNA over 18S nDNA. (F) Relative gene expression, by qPCR, of mitochondrial biogenesis regulatory genes in control vs senescent HepG2 and Huh7 cells. For all experiments the bar indicates values as mean ± SD of 3 independent experiments. P values were calculated by paired Student’s t test. *P ≤ .05, **P ≤ .01, ***P ≤ .001. The asterisk indicates heterochromatin. AV, autophagic vacuole; ER, endoplasmic reticulum; M, mitochondria; N, nucleus.