Figure 3

ERBB4 overexpression enhanced MSC survival through enhancing the mobility and anti-apoptosis by activating the PI3K/Akt pathway. (a) Surviving MSCe and MSC-ERBB4 were detected using anti-GFP antibody at 4 weeks post-MI. Representative images are shown. Quantification revealed that ERBB4 overexpression significantly improved MSC survival. Scale bar=200 μm. n=12 for each group. (b) The NRG1-ERBB4 pathway enhanced MSC mobility. (b1) Experiment setting: MSCe or MSC-ERBB4 were seeded in the upper chamber of a transwell unit, with NRG1 added to the lower chamber to serve as an attractor. (b2) After hypoxic exposure for 6 hours, MSCe and MSC-ERBB4 exhibited equivalent movements (b2i, NS); however, MSC-ERBB4 demonstrated more aggressive mobility in the presence of NRG1 than MSCe did (b2ii, P<0.05 versus MSCe). Scale bar=200 μm. (c) Annexin V/PI staining followed by flow cytometry were conducted to study the dynamic apoptotic rates of MSCe and MSC-ERBB4. Under hypoxia challenge, MSCe stayed incooperative to NRG1 (cii–cv), but the apoptotic rate of MSC-ERBB4 was attenuated by NRG1 in a dose-dependent manner under hypoxic condition (cvii–cx). The experiment was repeated three times and statistical analysis was performed (cxi). (d) Overexpressing ERBB4 in MSC reduced hypoxia-related apoptosis through the PI3K/Akt pathway. Additional NRG1 increased p-Akt accumulation in MSC-ERBB4 (dviii versus vii), but not in MSCe (dii versus di), and this effect could be partially blocked either by PI3K/Akt inhibitor LY294002 (dxii versus dviii), or anti-ER4 antibody (dxi versus dviii). The same trend was exhibited in Bcl-2 expression. The aforementioned western blotting was repeated three times and representative images are shown. Bcl-2, B-cell lymphoma 2; p-Akt, phosphorylated Akt; t-Akt, total Akt; anti-ERBB4, anti-ERBB4 antibody