NRCMs were seeded onto laminin-coated coverslips prior to infection with siRNA-control, siRNA-UCHL1, Ad-GFP, or Ad-UCHL1 for 24 hours, followed by PE stimulation for 24 hours. Immunofluorescence was performed as described previously (44). Briefly, the cells were washed with PBS and postfixed in 4% paraformaldehyde for 15 min at 20° to 23°C. After the cells were blocked in PBS containing 1% bovine serum albumin (BSA) and 0.2% Triton X-100 for 10 min, monoclonal antibody against sarcomeric α-actinin was added at dilutions of 1:200 for overnight incubation at 4°C. After washing with PBS three times for 5 min, the cells were then incubated with secondary antibody for 1 hour at 20° to 23°C, and the nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; 100 ng/ml) for 5 min. The cardiomyocyte surface area was depicted using ImageJ software (NIH, Bethesda, MD, USA), and the relative surface area was read with arbitrary units (the number of pixels) to evaluate hypertrophy.

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