Primary hepatocytes maintenance supplements

Primary hepatocytes were isolated by perfusing portal vein of 3 month old WT and Lrp6^mut/mut male mice and 6 month old Ldlr^−/− mice and Ldlr^−/−/Lrp6^mut/mut male mice on indicated diets (n=6) with collagenase (Type II collagenase, Gibco). Isolated hepatocytes were Percoll purified and cultured on collagen-coated tissue culture dishes in William’s medium supplemented Primary Hepatocytes Maintenance Supplements (Gibco) and kept in a humidified cell culture incubator at 37°C and 5% CO₂.

For LRP6 knockdown in HepG2 cells, cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% heat inactivated fetal bovine plasma and 1x penicillin-streptomycin at 37°C in a humidified O₂/CO₂ (19:1) atmosphere. Lentivirus particles containing LRP6 targeting shRNA (5′-CGGCGAATTGAAAGCAGTGAT-3′) was purchased (Santa Cruz Biotech) and was transduced into HepG2 cells. Transduced cells were selected using 1 μg/mL puromycin.
For Sp1 knockdown, isolated primary hepatocytes were cultured on collagen-coated tissue culture dishes in William’s medium supplemented with Primary Hepatocytes Maintenance Supplements (Gibco). Cells were infected with Sp1 shRNA (m) lentiviral Particles (sc-29488-V, Santa Cruz Biotech) using polybrene to increase the efficiency of infection.

Primary mice hepatocytes were cultured on collagen-coated tissue culture dishes in William’s medium supplemented with Primary Hepatocytes Maintenance Supplements (Gibco). Cells were serum starved overnight before the addition of 1 μM picropodophylin (PPP, Calbiochem) for 24 h or/and 100 nM recombinant human IGF1 (R&D Systems) for 15 min or 24 h. Whole cell lysates were analyzed by immunoblotting as described previously.