SFV enhances Puma mRNA and protein levels in MEFs and FDM cells, but mRNA increase is late and Bax/Bak-dependent.
(A) Quantitative/real time reverse transcriptase PCR (qRT-PCR) of Puma mRNA isolated from SV40 TAg WT and Bax/Bak-/- MEFs infected with 10 moi of SFV for 0, 1, 2, 4, 6, 8, 10, 14, 18 or 24 h (hpi). The mRNA values were normalized to the ribosomal housekeeping 18S gene and depicted as 2-∆∆Ct relative to mock cells (see Materials and Methods for details). Data are the means of at least three independent experiments ± SEM. The p values are the following: SFV-treated WT versus untreated: p = 0.005 for 6 h, p = 0.01 for 8 h, p = 0.008 for 10 h. SFV-treated Bax/Bak-/- versus untreated: not significant, n = 5. (B) Anti-Puma, anti-caspase-3 (pro-caspase-3 and cleaved caspase-3) and anti-PARP western blot analysis of total cell extracts of SV40 TAg WT MEFs infected with SFV for 0, 2, 6, 8, 10 or 18 h. Anti-actin as loading control.