Primary mouse hepatocytes were prepared by the collagen perfusion method. Eight- to 12-week-old male mice were anesthetized with ketamine/xylazine, and the vena cava was cannulated. The portal vein was cut immediately, and the liver was perfused with Earle’s balanced salt solution (without Ca2+/Mg2+ ; Thermo Fisher Scientific) supplemented with 0.5 mM EGTA. Subsequently, the buffer was replaced to Hanks’ balanced salt solution with Ca2+/Mg2+ (Biochrom) containing type I collagenase (100 U/ml) (Worthington). After sufficient digestion, the liver was excised and the gall bladder was removed. Cells were liberated into Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S), filtered through a 100-μm cell strainer, and centrifuged (50g, 3 min, 4°C). Afterward, Percoll (GE Healthcare) mixed with respective amounts of 10× PBS and culture medium was used to form a gradient that allowed enrichment of the hepatocyte fraction (50g, 10 min, 4°C), which was washed three times with culture medium (50g, 3 min, 4°C). Hepatocytes were then plated on collagen type I–coated 6- or 12-well plates (BD). After 4 to 6 hours, to allow attachment, the medium was replaced by fasting medium [DMEM supplemented with 0.2% FFA-free BSA (Sigma) and 1× P/S], and the cells were used the following day unless otherwise noted. For PKD3 phosphorylation studies, primary hepatocytes were incubated either with 1,2-dioctanoyl-sn-glycerol (Enzo Life Sciences) for 4, 6, and 8 hours or with oleic acid bound to albumin (Sigma) for 4 and 20 hours.

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