Humanin enhanced mitochondrial biogenesis in RPE cells. Nonpolarized RPE cells were treated with 150 μM tBH or 10 μg HN or 10 μg HN + 150 μM tBH for 24 hours and samples were analyzed for mitochondrial biogenesis by DNA copy number, TEM for mitochondrial number, and Western blot analysis for mtTFA. (A) The mtDNA copy numbers were determined by measuring the ratio of mtDNA to nuclear DNA (averaging the copy numbers of ND1/SLCO2B1 and ND5/SERPINA1) by real-time PCR. (B, C) Transmission electron microscopy was used to identify mitochondria (arrows) and quantitate mitochondrial number per cell in RPE cells treated with tBH and tBH plus HN. (D, E) Protein expression of mtTFA was measured by using Western blot analysis. The quantitative densitometry bar graph derived from three independent experiments showing significant upregulation of mtTFA with HN treatment. *P < 0.05, **P < 0.01. Scale bar: 0.2 μm.