PGC-1α silencing in RPE cells promotes mitochondrial dysfunction and oxidative stress. PGC-1α silencing in RPE cells promotes mitochondrial dysfunction.

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PGC-1α silencing in RPE cells promotes mitochondrial dysfunction and oxidative stress. PGC-1α silencing in RPE cells promotes mitochondrial dysfunction and oxidative stress. (A, B) Validation of PGC-1α silencing at the mRNA and protein levels by quantitative RT-PCR (A) and Western blotting (B) in ARPE-19 cells matured for 7 d (n = 3–4 per condition, respectively). (C) Evaluation of mitochondrial morphology using MitoTracker (red) and DAPI (nuclei, blue) co-staining showing prominent shape changes from tubular to “donut-blob” formation in shPGC1A cells (arrows). Scale bar is 10 μm. (D) Quantification of the mitochondrial dynamics genes FIS1 and MFN2 by qPCR (n = 3–4). (E) CSA quantification (n = 4). (F) OCR parameter measurements of shPGC1A and shCtrl ARPE-19 at day 7, 14, and 21 (n = 5). (G, H) Quantification of mitochondrial superoxide production using MitoSox Red (n = 5–7) (G) and cytoplasmic ROS content with DCFH-DA (n = 3–6) (H). (I) Relative expression of antioxidant enzyme genes—catalase (CAT), cytosolic superoxide dismutase (SOD1), mitochondrial thioredoxin (TXN2), and glutathione peroxidase (GPX)—analyzed by qPCR (n = 3–4). (J) Sirtuins (SIRT) enzymes 1 and 3 gene expression analysis by qPCR (n = 3–4). (K) PGC-1 isoform gene expression, PGC-1β, and PRC (n = 3). Error bars are means ± SEM. Data were analyzed by multiple unpaired t test comparisons using the Holm–Sidak method (A–D, F–K) or ANOVA followed by Tukey’s multiple comparison test (E). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 compared with their respective shCtrl at each time points. ###P ≤ 0.001; ####P ≤ 0.0001 compared with day 7 shCtrl. Mariana Aparecida Brunini Rosales et al. LSA 2019;2:e201800212 © 2019 Rosales et al.