Fig. 5.

cTnImAb1 upregulates the expression of ENO1 and induces cardiomyocyte apoptosis, while silencing ENO1 attenuates cTnImAb1–induced cardiomyocyte apoptosis.

a & b. cTnImAb1 treatment (0, 25, 50, or 100 μg/ml) for 48 h induced cardiomyocyte apoptosis in a dose-dependent manner as revealed by flow cytometry. a, representative images. b, quantification of 3 independent experiments. *p = .000 (ANOVA analysis).

c. cTnImAb1 treatment for 48 h at indicated doses increased Bax, cleaved Caspase3, and caspase8/p18 expression, and decreased Bcl2 expression in a dose-dependent manner as revealed by immunoblot analysis. β-actin served as a loading control. Shown are representative images of 3 independent experiments.

d&e. cTnImAb1 treatment (50 μg/ml) for indicated times induced cardiomyocyte apoptosis in a time-dependent manner as revealed by flow cytometry. d, representative images. e, quantification of 3 independent experiments, *p = .000 (ANOVA analysis).

f. cTnImAb1 treatment (50 μg/ml) for indicated times increased Bax, cleaved Caspase3, and caspase8/p18 expression, and decreased Bcl2 expression in a dose-dependent manner as revealed by immunoblot analysis. β-actin served as a loading control. Shown are representative images of 3 independent experiments.

g-i. cTnImAb1 treatment (50 μg/ml) for 48 h increased ENO1 expression of primary cultured cardiomyocytes (i) compared with mouse IgG (g) and cTnTmAb (h) treatments as revealed by immunohistochemistry. Bar = 50 μm.

j&k. cTnImAb1 treatment (50 μg/ml) for 48 h increased ENO1 expression compared with mouse IgG treatments as revealed by immunoblot analysis. β-actin served as a loading control. Shown are representative images (j) and quantification by ImageJ-mediated densitometry analysis (k) of 3 independent experiments. *p = .000 (t-test).

l&m. Silencing ENO1 attenuated cTnImAb1 (50 μg/ml)-induced cardiomyocyte apoptosis compared with mock-siRNA group. l, Annexin V-PI staining was used for flow cytometric analysis. Representative images are shown. m, Quantification of Annexin-V/PI staining from 3 independent experiments. 24 h *p = .008, 48 h *p = .000 (t-test).

n&o. RNAi-mediated silencing of Eno1 in primary cultured cardiomyocytes significantly reduced the cTnImAb1 (50 μg/ml)-induced cardiomyocyte apoptosis by increasing Bax expression and decreasing Bcl2 expression compared with mock-siRNA group. Representative immunoblot images are shown in n. ImageJ-based quantification of ENO1 (*ENO1-siRNA p = .000, Mock-siRNA p = .000), Bax (*ENO1-siRNA p = .000, Mock-siRNA p = .000), and Bcl2 (*ENO1-siRNA p = .003, Mock-siRNA p = .020) protein expression is shown in o. (ANOVA analysis).