Figure 9.

IGFBP-5 is localized on the cell surface and promotes myogenic differentiation by binding to and promoting IGF-II action. (A) 24 h after the induction of differentiation, C2C12 cells were fixed without (a) or with (b) membrane permeabilization and stained with an IGFBP-5 antibody. Bar, 10 μm. (B) 0 and 24 h after induction of differentiation, C2C12 cells were incubated with a high salt buffer to strip off membrane/ECM bound IGFBP-5. The released IGFBP-5 was detected by ligand blot. (C) C2C12 cells were switched to serum-free medium containing 300 ng/ml IGF-II or Des(1-6)IGF-II. 36 h later, the cells were subjected to MHC immunostaining and DAPI staining and the differentiation index determined as described in Materials and methods. Data are means ± SE of three independent experiments with duplicates. Group *, #, and • are significantly different from each other at P < 0.05. (D) Cells transfected with pSUPER or pSUPER-BP5 were switched to DM (containing 0.5% horse serum) without or with 50 ng/ml IGF-II, 50 ng/ml IGF-II + 210 ng/ml wild–type IGFBP-5, 210 ng/ml wild-type IGFBP-5, 50 ng/ml IGF-II + 210 ng/ml LBD-IGFBP-5, 210 ng/ml LBD-IGFBP-5, or 150 ng/ml IGF-II for 4 d. MHC levels were measured by Western immunoblot and quantified by densitometry. Values are expressed as relative to the IGFBP-5 siRNA group. Data are means ± SE of 3–4 independent experiments. Group * is significantly different from group # at P < 0.05.