Fig. 2. The combination of Hcy and Cu²⁺ induces p62-depedent autosis.

A Effect of CQ on cardiomyocyte viability with Hcy and Cu²⁺ for 24 h. The cells were pre-treated with 10 μM CQ 1 h before 800 μM Hcy and 20 μM CuCl₂ was added. Cell viability was analyzed by LDH release assay. Each bar represents the mean of three separate experiments, each measured in triplicate. B Reduced viability of H9c2 cells with Hcy and Cu²⁺ by Atg7 knockdown. H9c2 cells infected with Atg7 siRNA or non-targeting siRNA control (NC) was treated with Hcy and CuCl₂ for 24 h. Cell viability was analyzed by LDH release assay. Each bar represents the mean of three separate experiments, each measured in triplicate. C Ultrastructure of neonatal cardiomyocytes treated with or without Hcy and Cu²⁺ for 12 h (phase 1) and 20 h (phase 2). The representative transmission electron microscopic (TEM) images are shown. Lower panels: Details of autosis. Note the swollen perinuclear space (PNS), outer nuclear membrane (ONM, white arrow) and inner nuclear membrane (INM, white arrow) in phase 2 neonatal cardiomyocyte, and the presence of early (black arrows) and late (black star) autophagosomes in Hcy and Cu²⁺-treated cells but not control ones. N nucleus. D The effect of digoxin on cardiomyocyte viability with Hcy and Cu²⁺ for 24 h. H9c2 cells were pre-treated with 0.1 or 0.5 μM digoxin 1 h before 800 μM Hcy and 20 μM CuCl₂ was added. Cell death was determined by the loss of mitochondrial membrane potential detected by flow cytometry analysis (left), or assessed by the measurement of lactate dehydrogenase (LDH), released from dead cells (right). In the left panel, the numbers indicate the gating of a subpopulation of survival cells. Representative histograms of three separate experiments are shown. In the right panel, each bar represents the mean of three separate experiments, each measured in triplicate. E Expressions of p62 and 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 and rapamycin (RAPA) as positive control. F Expressions of p62 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. G Effect of p62 knockdown on LC3 expression in H9c2 cells with Hcy and Cu²⁺ incubation, assessed by western blot analysis. H9c2 cells infected with p62 siRNA or non-targeting siRNA control (NC) was treated with Hcy and CuCl₂ for 16 h. The representative western blot results are shown, with GAPDH expression as an internal control. H Effect of p62 knockdown on H9c2 cell viability with Hcy and Cu²⁺ incubation. The cells were transfected with p62 siRNA or non-targeting siRNA control (NC), followed by Hcy and CuCl₂ treatment for another 24 h. Cell viability was analyzed by LDH release assay. Each bar represents the mean of three separate experiments, each measured in triplicate. All the experiments above were performed three times. Ctrl, CQ, RAPA: see Fig. 1.