POS treatment decreases ROS level and upregulates mitochondrial biogenesis in ARPE-19 cells through PGC-1α.

<p><b>(A)</b> POS treatment upregulated mRNA levels of anti-oxidant enzymes including GPx1, GPx4, SOD1 and catalase. Mean ± SEM, n = 6 per group, two-tailed Student’s <i>t</i>-test, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001; ns, not significant. <b>(B)</b> POS treatment downregulated the Intracellular ROS levels evaluated by H₂DCFDA. Mean ± SEM, n = 6 per group, two-tailed Student’s <i>t</i>-test, **<i>P</i> < 0.01, ***<i>P</i> < 0.001; ns, not significant. <b>(C)</b> Knockdown Efficacy of PGC-1α siRNA was confirmed by mRNA levels measured by RT-PCR (Mean ± SEM, n = 6 per group, two-tailed Student’s <i>t-</i>test, ****<i>P</i> < 0.0001) and protein levels by western blot analysis (mean ± SEM, n = 3 per group, two-tailed Student’s <i>t-</i>test, **<i>P</i> < 0.01). <b>(D)</b> POS treatment decreased ROS level in cells transfected with control siRNA, but rather increased in those transfected with PGC-1α siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s <i>t</i>-test, ****<i>P</i> < 0.0001. <b>(E)</b> POS treatment upregulated relative mitochondrial DNA content. Mean ± SEM, n = 6 per group, two-tailed Student’s <i>t</i>-test, ****<i>P</i> < 0.0001. <b>(F)</b> Western blot analysis showed the increased protein level of mitochondrial marker prohibitin in cells treated with POS. Mean ± SEM, n = 3 per group, two-tailed Student’s <i>t</i>-test, *<i>P</i> < 0.05. <b>(G)</b> POS treatment upregulated mitochondrial complex I activity in cells transfected with control siRNA, (n = 8 per group, Tukey’s test, <i>P</i> < 0.001), but not in those transfected with PGC-1α siRNA (n = 8 per group, Tukey’s test, P > 0.05).