Pgc 1α sirna

Primary cultured periportal hepatocytes were individually transfected with PGC-1α, SIRT3 and SIRT5 siRNAs or scrambled control siRNA to silence target proteins. The hepatocytes were transfected with Lipofectamine 2000 (Invitrogen) using 150 pmol/well of the following siRNAs: SIRT3 siRNA (Production no. sc-61556, Santa Cruz); SIRT5 siRNA (Production no. sc-63027, Santa Cruz); PGC-1α siRNA (Production no. sc-38885, Santa Cruz) and scrambled (SCR, as control, Sigma-Aldrich) according to the manufacturer’s instructions. The siPGC-1α, siSIRT3 and siSIRT5 samples and the corresponding controls were detected after 24 h. Alternatively, to obtain PGC-1α overexpression hepatocytes, cells were cultured in Williams’ E medium (SigmaAldrich), containing either PGC-1α-expressing adenoviruses (PGC-1α-Ad) or LacZ-Ad (control) at MOI 0.1 according to Buler et al.19 (link). Preparation of the Ad-PGC-1α virus has been described previously30 (link). The cells were detected 48 h after infection.

CWR22Rv1 (22Rv1), PC3, C4-2B, MIAPACA-2, and HPAF-II cells were purchased (American Type Culture Collection). Cell lines were cultured in RPMI-1640 medium in 10% fetal bovine serum. For counting cells for proliferation, 25 000 cells/24 wells were treated with oligomycin 1 h before radiation. Cells were collected and counted using a hemocytometer 72 h post treatment using 5 wells per treatment. The Gammacell 40 Exactor (Best Theratronics, Ottawa, CA) was used for irradiation at indicated doses. 22Rv1 were silenced using pooled target-specific 19- to 25-nucleotide siRNAs by transfecting with either control siRNA-A (sc-37007, Santa Cruz Biotechnology), PGC-1α siRNA (sc-38884, Santa Cruz Biotechnology), or P53 siRNA (sc-29435, Santa Cruz Biotechnology). Cells were transfected using Lipofectamine 3000 Reagent (ThermoFisher) according to the manufacturer’s protocol.