Fig. 6: PDE4D knockout and rolipram alleviates apoptosis in vivo.

a Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the Apoe^−/−Pde4d^flox/flox and Apoe^−/−Pde4d^SMC−/− mice with or without 1000 ng kg⁻¹ min⁻¹ Ang II treatment and a high-fat diet (HFD) for 28 days. b Quantification of cleaved caspase-3 protein expression by immunoblotting in (a) normalized to caspase-3 protein (fold change versus control). *p < 0.05, ****p < 0.0001, two-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, Apoe^−/−Pde4d^flox/flox (n = 5) and Apoe^−/−Pde4d^SMC−/− (n = 5) with saline infusion, Apoe^−/−Pde4d^flox/flox (n = 5) and Apoe^−/−Pde4d^SMC−/− (n = 5) with Ang II infusion. c Quantification of cell apoptosis rates with TUNEL staining in Supplementary Fig. 13. A total of five random images per mouse were selected for TUNEL staining statistical analysis. Apoptotic cells examined by TUNEL staining were quantified as the ratio of apoptotic cells to total cells in five fields per mouse. *p < 0.05, **p < 0.01, Welch ANOVA with Dunnett’s T3 post hoc test, mean ± SEM, Apoe^−/−Pde4d^flox/flox (n = 5) and Apoe^−/−Pde4d^SMC−/− (n = 5) with saline infusion, Apoe^−/−Pde4d^flox/flox (n = 5) and Apoe^−/−Pde4d^SMC−/− (n = 5) with Ang II infusion. (d) Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the Apoe^−/− mice with or without Ang II/rolipram treatment. e Quantification of cleaved caspase-3 protein expression by immunoblotting in (d) normalized to caspase-3 protein (fold change versus control). *p < 0.05, **p < 0.01, two-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, vehicle (n = 5) and rolipram (n = 5) with saline infusion, vehicle (n = 5) and rolipram (n = 5) with Ang II infusion. f Quantification of cell apoptosis rates with TUNEL staining in Supplementary Fig. 14. A total of 5 random images per mouse were selected for TUNEL staining statistical analysis. Apoptotic cells examined by TUNEL staining were quantified as the ratio of apoptotic cells to total cells in five fields per mouse. **p < 0.01, Mann–Whitney test, mean ± SEM, vehicle (n = 5) and rolipram (n = 5) with saline infusion, vehicle (n = 5) and rolipram (n = 5) with Ang II infusion.