Fig. 5Figure 6
SIRT1 knockdown abolishes the protection of PDE4D silencing on ISO-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. A through F, NRVMs were transfected with PDE4D siRNA (siPDE4D) and SIRT1 shRNA or negative control siRNA (siNC) for 48 h followed by vehicle or ISO (10 μM) treatment for another 24 h. A, Immunofluorescence staining of α-actinin (red) and DAPI (blue) and quantification of myocytes area in NRVMs; n = 24 cells from 3 independent experiments. Scale bar, 20 μm. B, FACS analyses of ΔΨm in NRVMs. A loss of ΔΨm was demonstrated by the ratio of JC-1 aggregates to JC-1 monomers; n = 3 independent experiments. C, DCFH-DA staining and quantification of cellular ROS levels in NRVMs; n = 8 independent experiments. Scale bar, 50 μm. D, MitoSOX staining and quantification of mitochondrial ROS levels in NRVMs; n = 8 independent experiments. Scale bar, 10 μm. E , Representative immunofluorescence images of SIRT1 staining (red) and DAPI staining (blue) in NRV