Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Senescent Hepatocytes in Decompensated Liver Show Reduced UPR MT and Its Key Player, CLPP, Attenuates Senescence In Vitro

doi: 10.1016/j.jcmgh.2019.03.001

Figure Lengend Snippet: CLPP overexpression altered mitochondrial respiration in hepatoma cells. ( A ) Representative extracellular flux assay plots of HepG2 and Huh7 cells, expressing either GFP or CLPP, using the Seahorse XF Cell Mito Stress Test Kit. Respiration was monitored on sixth day following treatment with either Dox or vehicle (Con). The OCR was monitored at basal level in the absence of exogenous substrate, followed by sequential addition of oligomycin (1.0 μM), FCCP (0.50 μM for HepG2 and 0.25 μM for Huh7), and rotenone/antimycin A (0.5 μM). Points represent mean ± SD. Protein content of each well was used to normalize the OCR. ( B ) Bar graphs represent basal respiration in both HepG2 and Huh7 cells expressing either GFP or CLPP. ( C ) Bar graphs represents maximal respiration in both HepG2 and Huh7 cells expressing either GFP or CLPP. ( D ) Bar graphs represent adenosine triphosphate linked respiration in both HepG2 and Huh7 cells expressing either GFP or CLPP. ( E ) OCR of isolated mitochondria from GFP and CLPP overexpressing HepG2 and Huh7 cells as measured by oxygen microsensor (TBR1025 single-channel free radical analyzer). 2 mM Nicotinamide adenine dinucleotide was added (arrow) after 5 minutes and its oxidation was monitored every 30 seconds. ( F ) Energy phenotype analysis by plotting OCR vs extracellular acidification rate in CLPP and GFP overexpressing cells with or without Dox treatment. The bar graph represents value as mean ± SD and P values was calculated by Student’s t test. * P ≤ .05, ** P ≤ .001.

Article Snippet: The isolated mitochondrial fraction was resuspended in suspension buffer (HEPES [20 mM, pH 7.2], sucrose [0.3M], EDTA [1 mM], MgCl 2 [2 mM], KH 2 PO 4 [0.5 mM]) and the change in dissolved oxygen concentration was monitored every 30 seconds by using TBR1025 single-channel free radical analyzer (World Precision Instruments, Sarasota, FL).

Techniques: Over Expression, XF Assay, Expressing, Isolation