FIGURE 5.

LRP1/CRT co-receptor system is involved in inhibitory effects of adiponectin on DOX-induced myocyte apoptosis. A, detection of CRT on the cell surface of cardiac myocytes by flow cytometric analysis. Myocytes were incubated with anti-CRT antibody or control IgY for 60 min. B, anti-CRT antibody (ab) diminishes the binding of fluorescence-labeled adiponectin to cardiac myocytes. Myocytes were preincubated with anti-CRT antibody (200 μg/ml) or control IgY (200 μg/ml) for 60 min followed by incubation with FAM-labeled adiponectin (6 μg/ml) for 60 min. C, Western blot analysis for adiponectin-induced phosphorylation of Akt in the presence of anti-CRT antibody or control IgY. Myocytes were pretreated with anti-CRT antibody or control IgY for 60 min and treated with adiponectin (10 μg/ml) or vehicle for 16 h. Quantitative analysis of relative changes in phosphorylated Akt (P-Akt) is shown. Phosphorylation of Akt was normalized to the α-tubulin signal. D and E, effect of the anti-CRT antibody on adiponectin inhibition of myocyte apoptosis after DOX stimulation. Cells were pretreated with anti-CRT antibody or control IgY for 60 min and treated with adiponectin or vehicle for 24 h. Quantitative analysis of TUNEL-positive nuclei (D) and caspase-3 activity (E) is shown. F, the representative immunoblots of phosphorylated Akt (p-Akt) following treatment with adiponectin for 16 h in the presence of siRNA targeting CRT, LRP1, AdipoR1, or AdipoR2 or unrelated siRNA in cardiac myocytes (top). Bottom, quantitative analysis of relative changes in phosphorylated Akt. G and H, effect of knockdown of CRT, LRP1, AdipoR1, or AdipoR2 on adiponectin inhibition of TUNEL-positive myocytes (G) and caspase-3 activity (H) after DOX stimulation (n = 4–6).