Figure 7.

PCI34051 and Hdac8 or Ace1 knockdown regulate Rela and Nfkbia expression in cardiomyocytes. (a) mRNA levels of Hdac8, Ace1, Ace2, Rela, and Nfkbia in H9c2 cells transfected with pCMV vector or pCMV-Hdac8 were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) (n = 6 per group). (b) mRNA levels of Hdac8, Ace1, Ace2, Rela, and Nfkbia in H9c2 cells transfected with control short-interfering RNA (siRNA) or siRNA against Hdac8 (si-Hdac8) were evaluated using qRT-PCR (n = 12 per group). (c, d) Representative immunoblots and quantification of Ace1, Ace2, Hdac8, Rela, and Nfkbia levels in pCMV6-Ace1-transfected H9c2 cells (n = 6 per group). Actb was used as a loading control. (e) mRNA levels of Ace1, Ace2, Hdac8, Rela, and Nfkbia in H9c2 cells transfected with control siRNA or si-Ace1 were evaluated using qRT-PCR (n = 12 per group). (f, g) The mRNA levels of Rela and Nfkbia in H9c2 cells transfected with control siRNA or si-Hdac8 and treated with isoproterenol (10 μM) for 9 h were measured using qRT-PCR (n = 6 per group). (h, i) The expression levels of Hdac8, Ace1, Ace2, Rela, and Nfkbia in H9c2 cells transfected with pCMV or pCMV-Hdac8 and control siRNA or si-Ace1; representative immunoblots and quantification of protein levels are shown (n = 6 per group). Actb was used as a loading control. (j, k) H9c2 cells were incubated with TNFα (50 ng/mL) for 1 h and cultured in the presence of vehicle or PCI34051 (10 μM) for 5 h. The nuclear and cytoplasmic fractions were obtained as described in Materials and Methods. Representative western blots and quantification of protein levels (n = 4 per group). Actb and LmnB were used as loading controls for the cytoplasmic and nuclear fractions, respectively.