Rat tail tendon collagen

HEK 293T and HepG2 cells were purchased from ATCC, cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% l-glutamine and checked for mycoplasma by PCR or ELISA. For luciferase reporter assay, HEK 293T cells were transfected using 50 ng of Per1-Luc and 20 ng of β-gal, together with 100 ng of Bmal1 and Clock plasmids per well. Co-transfections were performed with a constant amount of DNA by adding 100 ng of the empty vector pcDNA3 (Invitrogen) and luciferase activity was measured. Mouse primary hepatocytes were prepared, cultured and infected with adenoviruses. Briefly, the hepatocytes were isolated by using collagenase (Sigma) and plated on dishes coated with rat tail tendon collagen (Sigma) according to manufacturers' instructions. Before experiments, all the cells were synchronized by 12 h serum starvation. For experiments with LY-294002 (Sigma, LY) or LMB (Cell Signaling Technology, LMB), hepatocytes were pre-treated with LY (10 μM) or LMB (20 ng ml⁻¹) for 2 or 5 h, before incubation with insulin (50 nM) for indicated times.

HEK293T cells were purchased from American Type Culture Collection (ATCC). Mouse primary hepatocytes were isolated by using collagenase (Sigma) and plated on dishes pre-coated with rat tail tendon collagen (Sigma) according to manufacturers’ instructions. HEK293T cells and primary hepatocytes were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and medium 199 (M199), respectively. Complete medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. HEK293T cells were transfected by using PEI (Sigma) reagent according to manufacturers’ instructions. Briefly, plasmid (ug) and PEI reagent (1 ug/uL) were mixed in a ratio of 1:3, and then incubated for 15 min at room temperature. Medium was changed into serum-free DMEM, and then PEI/Plasmid transfection complexes were added into wells. Cells were lysed for western blot or luciferase activity analysis after 24 hours post-transfection. Primary hepatocytes were synchronized by serum starvation for overnight and then infected by adenovirus as specified.