Fig. 1: Hcy and Cu²⁺ induce both the caspase-3-dependent apoptosis and autophagy in neonatal cardiomyocytes.

A Effect of Hcy and Cu²⁺ on cardiomyocyte viability. The cells were incubated with 800 μM Hcy and 20 μM CuCl₂ alone or in combination for 24 h. Cell viability was analyzed by LDH release assay. Each bar represents the mean of three separate experiments, each measured in triplicate. B Expressions of full-length PARP-1 (f-PARP-1), cleaved PARP-1 (c-PARP-1), and cleaved caspase-3 (c-Casp-3) in cardiomyocytes with Hcy and Cu²⁺ incubation at given time points, as assessed by western blot analysis. The representative western blot results are shown, with GAPDH expression as an internal control. C Effect of zVAD-fmk on cardiomyocyte viability with Hcy and Cu²⁺ for 24 h. The cells were preincubated with 800 μM Hcy and 20 μM CuCl₂ for 24 h before 20 μM zVAD-fmk was added. Cell viability was measured using annexin V/PI double staining. Representative dot plots of a cardiomyocyte sample are shown, with numbers indicating the percentage of viable cells (annexin V/PI double negative). D Expressions of LC3 in cardiomyocytes with Hcy and Cu²⁺ incubation at given time points, assessed by western blot analysis. The representative western blot results are shown, with GAPDH expression as an internal control. E Expressions of LC3 in cardiomyocytes with Hcy and Cu²⁺ incubation with or without CQ at given time points, assessed by western blot analysis. The representative western blot results are shown, with GAPDH expression as an internal control. F Representative images of acridine orange-stained cardiomyocytes with Hcy and Cu²⁺ incubation for 12 h. G Quantification of acridine orange staining using flow cytometry. Representative dot plots of three separate experiments are shown (left panel). The bar (right panel) represents the mean of three separate experiments, each measured the proportion of the events above the threshold with R/GFIR-T. All the experiments above were performed three times. Ctrl control, f-PARP-1 full-length PARP-1, c-PARP-1 cleaved PARP-1, c-Casp-3 cleaved caspase-3, CQ Chloroquine, RAPA rapamycin, R/GFIR red-to-green fluorescence intensity ratio.