Fig 1.
SGs exist in tachypaced HL-1 cells and atrial myocytes.
(A,B) Endogenous SGs was probed with anti-PABP1 antibody (A) and anti-G3BP-1 antibody (B) in paced HL-1 cells at different time points. (C) Cardiomyocytes isolated from newborn SD rats were transfected with green fluorescent pEGFP-C1-G3BP1 plasmid and then paced. (D) The presence of SGs in atrial myocytes without and with tachypacing was confirmed by electron microscopy (scale bar 30 μm, ↑indicates the nucleus and ↑↑indicates the SGs).
Fig 2.
SGs were induced by pacing and G3BP1 overexpression in the stable HL-1 cell line.
(A) Immunofluorescence was used to visualize SGs, which were indicated by G3BP1 protein (green) and endogenous PABP-1 protein (red), as well as their colocalization. Ctrl group: HL-1 cells; Paced group: cells stably expressing control plasmid that were paced for 6 h; and P+G3BP1 group: cells stably expressing G3BP1 that were paced for 6 h (scale bar is 100 μm). (B) Percentage of cells containing SGs (%), n = 5. *P < 0.05 compared with the ctrl group, #P < 0.01 compared with the paced group. (C) Number of SGs in the 3 groups, n = 6. *P < 0.01 compared with the Ctrl group, #P < 0.05 compared with the Paced group. (D) The expression of G3BP1 protein as detected by western blot was quantified; n = 4, *P < 0.01 compared with the pEGFP-LV group. All data are expressed as the mean ± SEM.
Fig 3.
Effect of exogenous G3BP1 on ROS production and calcium overload in the paced stable HL-1 cell line.
Flow cytometry was used to analyze the level of ROS (A) and the amount of Ca2+ (C) in HL-1 cells, n = 6. (B,D) Quantification of relative ROS and Ca2+ levels; n = 6. Data are expressed as the mean ± SEM. Ctrl: HL-1 cells; Paced: HL-1 cells paced for 6 h; P+Ctrl: cell lines stably expressing control plasmid that were paced for 6 h; and P+G3BP1: cell lines stably expressing G3BP1 that were paced for 6 h. *P < 0.01 compared with the Ctrl group; # P < 0.05 compared with the P+Ctrl group.
Fig 4.
G3BP1 prevent AngII-induced cardiac fibroblast proliferation and collagen synthesis.
A, Proteins were detected by western blot. B-E, Data are expressed as the mean ± SEM (n = 6). F. The CCK8 method was used to analyze the proliferation of cardiac fibroblasts induced by AngII and/or transient transfection of G3BP1 cDNA. *P < 0.05 compared with the control group; #P < 0.05 compared with the AngII+Ctrl group. **P < 0.01 compared with the control group; ##P < 0.01 compared with the AngII+Ctrl group.
Fig 5.
Protein synthesis in paced HL-1 cells.
A, Puromycin was incorporated into HL-1 cells and proteins were detected by western blot. B, Puromycin was incorporated into HL-1 cells and protein synthesis in single cells was analyzed by immunofluorescence. Puromycin was indicated by green fluorescence and G3BP1 was indicated by red fluorescence signals. C,D The amount of protein is expressed as the mean ± SEM (n = 6). *P < 0.01 compared with the control group; #P < 0.05 compared with the control group. E, The ratio of protein to DNA (n = 6). F, Mean fluorescence intensity in HL-1 cells was shown as mean ± SEM (n = 6). *P < 0.01 compared with the cells without SG, #P < 0.01 compared with the cells containing SG.