Recombinant high‑mobility group box 1 induces cardiomyocyte hypertrophy by regulating the 14‑3‑3η, PI3K and nuclear factor of activated T cells signaling pathways

Figures

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  Figure 1.. rHMGB1 induces NMC hypertrophy. rHMGB1 significantly increased (A) the protein content per cell, (B) ANP protein levels and (C) the mRNA levels of ANP and BNP in NMCs (n=3). Data are presented as the mean ± standard error of the mean for continuous variables. One-way ANOVA was used to assess differences among different groups. (D) Representative confocal images from the Ctrl and rHMGB1 groups (scale bar, 20 µm). Cells were stained with DAPI (blue signal) and α-actinin (green signal) to identify nuclei and cardiomyocytes, respectively. rHMGB1, recombinant high-mobility group box 1; NMC, neonatal mouse cardiomyocytes; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; Ctrl, control; DAPI, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. *P<0.05, **P<0.01 vs. Ctrl group.

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  Figure 2.. Effects of rHMGB1 on the protein content per cell and the protein levels of 14-3-3, ANP and p-Akt in neonatal mouse cardiomyocytes. (A) Panel represents western blot results from the different treatment groups. The other panels represent the semi-quantitative analysis of (B) 14-3-3η, (C) protein per cell, (D) pan-13-3-3, (E) ANP and (F) p-Akt/Akt from the four treatment groups. Each experiment was repeated three times (n=9). Data are presented as the mean ± SEM for continuous variables. One-way ANOVA was used to assess differences among the groups. rHMGB1, recombinant high-mobility group box 1; ANP, atrial natriuretic peptide; p-Akt, phosphorylated-Akt; Wort, wortmannin; Ctrl, control. *P<0.05 vs. Ctrl group; ^P<0.05 vs. rHMGB1 group; #P<0.05 vs. Wort group.

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  Figure 3.. rHMGB1 induces NFAT3 translocation to the nucleus. Representative confocal images of NFAT3 localization in neonatal mouse cardiomyocytes in different treatment groups (scale bar, 15 µm). Red indicates NFAT3 protein, blue indicates DAPI-stained nuclei and green indicates α-actinin. Each experiment was repeated four times. rHMGB1, recombinant high-mobility group box 1; NFAT3, nuclear factor of activated T cells 3; DAPI, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; Wort, wortmannin; Ctrl, control.

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  Figure 4.. rHMGB1 induces NFAT3 translocation to the nucleus. (A) Representative western blot results from cytosol or nuclear extracts from different treatment groups. (B) Semi-quantitative analysis of NFAT3 in cytosol extracts, with β-tubulin serving as the loading control. (C) Semi-quantitative analysis of NFAT3 in nuclear extracts, with histone serving as a loading control. The three independent neonatal mouse cardiomyocyte cultures were used in each experiment (n=3). One-way ANOVA was used to assess differences among different groups. rHMGB1, recombinant high-mobility group box 1; NFAT3, nuclear factor of activated T cells 3; Wort, wortmannin; Ctrl, control. *P<0.05 vs. Ctrl group; ^P<0.05 vs. rHMGB1 group; #P<0.05 vs. Wort group.

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  Figure 5.. rHMGB1 has no effect on the apoptosis rate of neonatal mouse cardiomyocytes assessed using a TUNEL assay. (A) Fluorescence microscopic images of TUNEL-positive nuclei. Red indicates TUNEL-positive nuclei and blue indicates total nuclei (scale bar, 200 µm). The experiment was repeated (five independent experiments were performed). TUNEL-positive nuclei were counted in four fields for each experiment under a fluorescent microscope (n=12). (B) Data analysis of the number of TUNEL-positive nuclei from the different groups. One-way ANOVA was used to assess differences among the different groups. rHMGB1, recombinant high-mobility group box 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end-labeling; Wort, wortmannin; Ctrl, control; DAPI, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine.

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  Figure 6.. Proposed effects of rHMGB1 on cardiomyocyte hypertrophy. rHMGB1 may induce cardiomyocyte hypertrophy by regulating the 14-3-3/PI3K/Akt/NFAT3 signaling pathway. The red solid arrows and the solid T-shaped lines indicate the stimulatory and inhibitory effects, respectively. The dashed line indicates the probable effect. rHMGB1, recombinant high-mobility group box 1; NFAT3, nuclear factor of activated T cells 3.