Figure 7.

Effect of parkin depletion on hypertrophy, oxidative stress, mitochondrial biogenesis and mitophagy in neonatal rat cardiomyocytes (NCMs). (a) Cell index quantification by RTCA analysis in NCMs transfected with the siRNA specifically targeting rat parkin mRNA (si prk, red line) or the non-targeted siRNA (si NT, black line) as control. Cell index was recorded every 15 min (n = 2 independent isolations, in quadruplicate). (b) Hypertrophy was quantified by immunofluorescence of alpha-actinin (red) and nuclei (blue) and quantification of cell area (µm²) (from 3 independent experiments and at least 178 cells). (c) Mitochondrial superoxide anion was quantified by fluorescence quantification of mitoSOX (red) (from 3 independent experiments and at least 249 cells (d) Mitochondrial membrane potential was quantified by fluorescence quantification of JC-1 dye (aggregates (red) and monomer (green)) (from 3 independent experiments and at least 257 cells). (e) Mitophagy was quantified in NCMs transfected with the si prk (+) or si NT (−) as control. by Western blot of Parkin, ubiquitinated proteins, Beclin-1 and LC3II/LC3I ratio. Data were normalized to HPRT for RNA and actin for protein. Only significant p values are indicated from at least 3 independent experiments. Images were selected to represent the mean values of each condition.