FIGURE 3.

The in situ proximity ligation assay showed that MID-1 disrupts the molecular association of MG53 with IRS-1. The chemical structural formula of MID-1: 4-ethoxy-N-(5-nitrothiazol-2-yl)benzamide (A). 4-Day-differentiated C2C12 myotubes were incubated with different combinations of 2.5 μm MG132 and 5 μm MID-1 for 4 h. The molecular association of MG53 with IRS-1 and FAK was determined by in situ proximity ligation assay (B). The fluorescent signals for MG53-IRS-1 (C) and MG53-FAK (D) interactions were statistically determined from 12 different image fields. t test; **, p < 0.01. The relative luciferase activity and cell survival rate of CLUC-C14A- and NLUC-IRS-1-overexpressing HEK 293 clone 1 were determined after treating the cells with different concentrations of MID-1 for 12 h. The relative luciferase activity (E) and MTT activity (F) were presented as percentages of the control without MID-1 treatment, respectively.