Figure 5.

FGF21 overexpression activated FGFR1 and ERK1/2 in H9c2 cells, and the inhibitors of FGFR1 and ERK1/2 attenuate cardioprotective effect of FGF21 on ER stress injury. (A) Subsequent to transfection with the pcDNA4-FGF21 plasmid, the expression levels of FGF21, p-FGFR1, FGFR1, p-ERK1/2 and ERK1/2 were analyzed by western blotting and their relative protein expression levels were compared. ^*P<0.05 or ^**P<0.01 vs. control, ^&P<0.05 or ^&&P<0.01 vs. TM group, ^#P<0.05 or ^##P<0.01 vs. pcDNA4 + TM group. (B) H9c2 cells were pretreated with the specific FGFR1 inhibitor, PD166866 (100 nM) or ERK1/2 inhibitor, PD98059 (20 µM) for 1 h, then transfected with pcDNA4-FGF21 plasmids for 48 h. TM (10 µM) was added to the media and incubated at 37°C for 24 h. The cell viability was evaluated by Cell Counting Kit-8 assay. ^*P<0.05 vs. TM group, ^#P<0.05 vs. pcDNA4-FGF21 + TM group. (C) Fluorescence microscopy of TUNEL staining of H9c2 cells. The cell nucleus was stained with DAPI (blue). Scale bar, 50 µm. The percentage of apoptotic cells in each group was calculated by counting the condensed nuclei. ^**P<0.05 vs. TM group, ^##P<0.05 vs. pcDNA4-FGF21 + TM group. FGF21, fibroblast growth factor 21; FGFR1, fibroblast growth factor receptor 1; ERK, extracellular signal-regulated kinases; TM, tunicamycin; ER, endoplasmic reticulum; p, phosphorylated; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling; c, control.