Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 3

Research Article

Synergistic Effects of Cilostazol and Probucol on ER Stress-Induced Hepatic Steatosis via Heme Oxygenase-1-Dependent Activation of Mitochondrial Biogenesis

Figure 3

Combinatorial treatment of cilostazol (CZ) and probucol (PB) ameliorates tunicamycin- (TM-) induced mitochondria dysfunction. Primary hepatocytes were pretreated with probucol (0.1 μM) and cilostazol (3 μM) individually as well as with their combination for 30 min followed by stimulation of tunicamycin (10 μg/mL) for 18 h. Mitochondrial mass was assessed by using MitoTracker Red (red). Nuclei were stained with Hoechst dye (blue). Images of fluorescence were analyzed by confocal microscopy (a). The relative mtDNA content was measured by real-time PCR. mtDNA content was normalized by nDNA content (b). ATP production was measured in primary hepatocytes isolated from HO-1 WT and KO mice (c). The expression of HO-1 and mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 as well as related proteins PGC-1α, COX III, and COX IV was measured by RT-PCR (d) and Western blot analysis (e). RT-PCR was also performed in HO-1 KO mice to detect mitochondrial biogenesis-related genes PGC-1α, TFAM, and NRF-1 (f). HepG2 cells were transfected with scRNA and siHO-1 for 24 h. Then cells were pretreated with probucol (0.1 μM) and cilostazol (3 μM) individually or combinatorially for 30 min followed by stimulation of tunicamycin (10 μg/mL) for another 18 h. RT-PCR was performed to detect HO-1, PGC-1α, TFAM, and NRF-1 mRNA levels (g). Bar graphs, lower panels of (d) and (e), as well as lower panel of (f), are summary data of normalized densitometry ratios. Quantitative data are expressed as means ± SE; . , , and versus cells without treatment; and versus cells treated with tunicamycin; and versus cells treated with probucol and tunicamycin; , , and versus cells treated with cilostazol and tunicamycin.
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