Fig. 2.

Effect of hypoxia in changing IGF actions is HIF-1–dependent. (A) Knockdown of HIF-1α by siRNA. Two days after transfection with 2 μg pSUPER (c) or pSUPER HIF-1α plasmid (1 and 2), cells were subjected to hypoxia for 8 h. Cells treated with CoCl₂ were used as positive control (PC). Nuclear fraction was prepared from normoxia and hypoxia groups and analyzed by Western blot. (B) HIF-1α siRNA abolishes HRE-dependent expression. C2C12 myoblasts were transfected with pSUPER (control) or pSUPER HIF-1α together with the HIF-1 reporter gene, p2.1, or the control p2.4 gene (which has a mutated HRE) and were grown under normoxia or hypoxia. At 24 h later, cells were lysed and used for luciferase activity assay. Data are means ± SE, n = 4. (C and D) Knockdown of HIF-1α abolishes mitogenic action of IGF-II but restores myogenic response to IGF-II under hypoxia. Cells transfected with pSUPER or pSUPER HIF-1α plasmid were cultured in differentiation medium with or without IGF-II (300 ng/mL) under normoxic (N) or hypoxic (H) conditions. Differentiation index (C) and total cell number (D) were determined. Data are mean ± SE, n = 4. (E and F) Overexpression of HIF-1α abolishes myogenic response to IGF-II under normoxia. Cells transfected with control or HIF-1α expression plasmid were cultured in differentiation medium with or without IGF-II (300 ng/mL) under normoxic or hypoxic conditions. Differentiation index and total cell number were determined. Data are mean ± SE, n = 3.