Figure 1.

SPRC induces STAT3 phosphorylation and nuclear translocation via gp130 in cardiomyocytes. H9c2 cardiac myocytes were treated either (a) with 30 μM SPRC for the indicated durations or (b) with increasing concentrations of SPRC for 10 min. Cell lysates were collected and subjected to western blot for evaluating phospho-gp130 and phospho-STAT3. (c) H9c2 cells were treated with SPRC (30 μM) for 10 min. Cells lysates were immunoprecipitated with gp130 or JAK2 antibody, and the bound proteins were fractionated on SDS-PAGE gel, then analyzed by western blot with antibodies indicated. (d) Cells were incubated with SPRC (30 μM) for 10 min, and then double-immunostained for gp130 and STAT3 and the nuclei were visualized by DAPI staining. Scale bar, 50 μm. (e) Cells were pretreated with 2 μM SC144 (gp130 inhibitor) or 10 μM WP1066 (STAT3 inhibitor) for 1 h, followed by SPRC stimulation. (f) Cells were incubated with SPRC after gp130 silencing, which was conducted by siRNA transfection for 48 h. (g) Cells were treated with SPRC for the indicated durations, or pretreated with/without SC144 for 1 h before SPRC stimulation for 10 min. Levels of STAT3 in the cytosol and nucleus were analyzed by western blot. GAPDH and Lamin B1 were used as loading control for cytosolic and nuclear proteins, respectively. (h) STAT3 nuclear translocation after 10 min of SPRC treatment was detected by confocal microscopy. Scale bars, 50 μm. Values are presented as mean±S.D. from n=3 replicates. ^#P<0.05, ^##P<0.01, ^###P<0.001 compared with the control group; ^**P<0.01 compared with the SPRC-treated group. NS, nonsignificant (P>0.05)