MiR-494 induced HIF-1α and HO-1 expression by activating PI3K/Akt pathway. (A) Cells were transfected with miR-494 mimic or miR-negative control under normoxia as described above. After transfection, cells were treated with or without 30 μM of LY294002 (PI3K inhibitor, block the PI3K/Akt pathway) for 8 hours. mRNA levels of HIF-1α and HO-1 were analyzed by real-time RT-PCR after transfection for 48 hours. (B) The expression of PTEN, p-Akt, Akt, HIF-1α, HO-1 and β-Tubulin were analyzed by western blot after 48 hours for transfection. Left: Western blot analysis for proteins. The signal indicated by white arrow is HIF-1α. Right: Densitometric analysis of the western blot in left. (C-D) Transfected cells were continuously treated with or without 30 μM of LY294002 for 16 hours prior to hypoxia (1% O2, 5% CO2 in a 37°C incubator). Eight hours later, cells were harvested for RNA and protein, real-time RT-PCR (C) and western blot (D. Left: Western blot analysis for proteins. Right: Densitometric analysis using Quantity one software in left) were done. Control indicates miR-negative control. MiR-494 + LY indicates treatment with LY294002 in transfected cells. Data were normalized by the level of β-Tubulin expression except p-Akt in panel B and panel D. The data of p-Akt was normalized by the level of Akt expression. The data were presented as the means ± SD. Columns, mean of three independent experiments; bars, SD; *p < 0.05.