Modulation of Hepatocyte Mitochondrial Regulators

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.

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Li L., Zhang P., Bao Z., Wang T., Liu S, & Huang F. (2016). PGC-1α Promotes Ureagenesis in Mouse Periportal Hepatocytes through SIRT3 and SIRT5 in Response to Glucagon. Scientific Reports, 6, 24156.

Publication 2016

Lipofectamine 2000 SIRT3 siRNA PGC-1α siRNA Williams’ E medium

Adenoviruses Cells Cultured medium Hepatocytes Infection Lacz Lipofectamine 2000 Pgc 1α Proteins target Sirna Sirt3 Virus

Corresponding Organization :

Other organizations : Huazhong Agricultural University

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Variable analysis

independent variables

PGC-1α siRNA
SIRT3 siRNA
SIRT5 siRNA
Scrambled (SCR) siRNA (control)
PGC-1α-expressing adenoviruses (PGC-1α-Ad)
LacZ-Ad (control)

dependent variables

Silencing of target proteins (PGC-1α, SIRT3, SIRT5)
PGC-1α overexpression

control variables

Primary cultured periportal hepatocytes
Lipofectamine 2000 transfection reagent
Williams' E medium

controls

Scrambled (SCR) siRNA (negative control)
LacZ-Ad (negative control)

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