3h glycerol

Sucrose density gradient centrifugation were used to separate IM and OM fractions as previously described [11 , 21 (link)]. For each strain, 30 mL cultures were grown to mid-log phase labeled with either 2 μCi/mL of [³H]-glycerol (Perkin-Elmer) or 2.5 μCi/mL of ³²P_i (Perkin-Elmer). Cells were harvested at 5,000 x g for 10 minutes and where appropriate, the supernatant saved for collection of shed OM material. The resulting cell pellet was suspended in 10 mM Bis-Tris pH 8.0 and centrifuged again. Cells were then resuspended in 6 mL of 10 mM Bis-Tris pH 8.0, 20% sucrose (w/w) and lysed by single passage through a mechanical cell press at 8,000 psi. Unbroken cells were removed by centrifugation at 5,000 x g for 10 minutes. Cell lysate was then layered on top of a two-step gradient consisting of 1.5 mL of 65% sucrose (w/w) and 5 mL of 40% sucrose (w/w). Samples were centrifuged at ~100,000 x g 16 hours, 0.8 mL fractions were then collected from the top.

Huh-7 cells were incubated in DMEM supplemented with 10% foetal calf serum and PSG containing 0.4 µCi per well of [¹⁴C]oleic acid for 24 hours, followed by lipid extraction and separation by TLC in hexane/diethyl ether/acetic acid (70/30/1, v/v). Radioactivity of TG fraction was measured.
HepG2 cells were incubated with MEM w/o FBS +6 µCi/mL [³H]glycerol (PerkinElmer) +1.5 mM glycerol (Sigma-Aldrich)+1% BSA (Sigma-Aldrich) for 5 hour. Cells were collected and washed with PBS to remove the excess of medium. Lipids were extracted adding 3 mL of chloroform:methanol (2:1 v/v) (Sigma-Aldrich) and 1 mL of acidified solution (17 mM NaCl, 1 mM H₂SO₄) (Sigma-Aldrich). Samples were centrifuged at 3000 rpm for 10 min. The lower organic phase was saved and dried under a gentle nitrogen stream. Lipids were reconstituted in 50 µL of chloroform (Sigma-Aldrich), and separated by one-dimensional TLC using TLC silica gel plates (Merck-Millipore). Triolein (Sigma-Aldrich) was used as a marker. Petroleum ether:diethyl ether:acetic acid (40:60:1, vol/vol) (Sigma-Aldrich) was used as the mobile phase. The area of TLC silica gel corresponding to the newly synthesised radio-labelled TG were cut out. The rate of released radio-labelled TG was measured, using liquid scintillation counting (PerkinElmer). Data were normalised for number of cells.

De novo triglyceride synthesis was analysed using ³H-glycerol^{56 (link)}. Briefly, McA-RH7777 and Huh7 cells were seeded in triplicate in six-well plates. Twenty-four hours after seeding, cells were transfected with Psd3 siRNA or SCR siRNA for 48 h in regular medium (supplementary material). Forty-eight hours after transfection, cells were incubated with ³H-glycerol (PerkinElmer) plus OA for 15, 30 or 60 min. Cell lysates were collected and lipids were extracted (supplementary material)^{57 (link)}, separated and quantified by thin-layer chromatography. Spots corresponding to triglycerides were visualized with iodine vapour and added to vials with scintillation fluid. Radioactivity was measured using a scintillation counter as disintegrations per min.