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. 2022 Dec 30;132(2):208–222. doi: 10.1161/CIRCRESAHA.122.321153

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© 2023 The Authors.

Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 7. — p53 mediates the proapoptotic effects of ASPP1. A, The efficiency of small interfering RNA (siRNA) of p53 in neonatal mouse ventricular cardiomyocytes (NMVCs) by Western blot (Mann-Whitney U test); n=5. B, LDH release after cotransfection of siRNA of p53 and ASPP1 plasmid (1-way ANOVA, followed by Tukey post hoc multicomparisons test); n=9. C, Apoptosis index of NMVCs detected by TUNEL (TdT-mediated dUTP nick end labeling) assay (Kruskal-Wallis, followed by false discovery rate [FDR] method of Benjamini and Hochberg test); n=5. Scale bar=50 μm. Green, TUNEL- positive cells; blue, DAPI. D, Caspase-3 activity in NMVCs by ELISA assay (1-way ANOVA, followed by Tukey post hoc multicomparisons test); n=6. E and F, The protein levels of Bcl2 and Bax detected by Western blot (1-way ANOVA, followed by Tukey post hoc multicomparisons test); n=6. G, Validation of p53-NT and p53-CT by Western blot. Flag-p53-NT, N-terminal fragment containing binding domain for ASPP1 without nuclear localizing signal (NLS, 1–288 aa) and Flag-p53-CT, C-terminal fragment containing NLS of p53, which is responsible for its nuclear localization (310-381 aa); n=5. H, Celluar distribution of Flag-p53-NT and Flag-p53-CT by immunofluorescent staining; n=5. Scale bar=20 μm. I, Effects of Flag-p53-NT and Flag-p53-CT on ASPP1 and p53 nuclear translocation in NMVCs; n=5. Scale bar=20 μm. J, LDH level in culture medium after treated with Flag-p53-NT or Flag-p53-CT (1-way ANOVA, followed by Tukey post hoc multicomparisons test); n=6. K, Caspase-3 activity in NMVCs after cotransfection of Flag-p53-NT or Flag-p53-CT and ASPP1 plasmid by ELISA assay (1-way ANOVA, followed by Tukey post hoc multicomparisons test); n=6. L and M, The protein levels of Bcl2 (L) and Bax (M) after cotransfection of Flag-p53-NT or Flag-p53-CT and ASPP1 plasmid detected by Western blot (1-way ANOVA, followed by Tukey post hoc multicomparisons test); n=6. ASPP1 indicates apoptosis stimulating of p53 protein 1; Bax, Bcl2-associated X; DAPI, 4′,6-diamidino-2-phenylindole; H/R, hypoxia/reoxygenation; and LDH, lactate dehydrogenase.