Recovery of mitochondrial biogenesis and neuroprotection by humanin treatment is PI3K/AKT signaling-dependent. (A, B) Western blot analysis to measure PI3K/AKT, ERK, and JNK activity from the striatum brain tissues of MPTP-treated PD mice with HN treatment (0.1 mg/kg, 5 mg/kg) as described above. (C) Differentiated PC12 cells were pre-treated with MK2206 (AKT inhibitor, 1 μM), wortmannin (PI3K inhibitor, 1 μM), or U0126 (ERK inhibitor, 10 μM) 2 h before treatment with humanin (HN, 10 μM) prior to MPP+ challenge for 24 h (1 mM). Drug effects on these cellular signaling pathways were confirmed by western blot analysis. (D, E, F) Phase-contrast pictures (D), MTT assay (E), and mitochondrial mass evaluation (F) of differentiated PC12 cells with pre-treatment of MK2206, wortmannin, and U0126 in HN treated MPP+-challenged cells. (G, H) Western blot analysis of the genes involved in mitochondrial biogenesis with pre-treatment of wormannin in humanin treated MPP+-challenged differentiated PC12 cells. Data are expressed as means ± SEM. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). HN: humanin. MK: MK2206, WO: wortmannin, U0: U0126.