Fig. 2.

CPT activity and the de novo biosynthesis of choline PL from radiolabeled choline in WT, CPT1-KO, CEPT1-KO, and DKO cells. A and B: CPT activity in cell lysates from WT, CPT1-KO, CEPT1-KO, and DKO HEK293 cells. Enzymatic activity was measured using radiolabeled CDP-choline and 18:1 DAG as substrates at 37°C for the time indicated. The synthesized radioactive PC was analyzed by TLC (A) and quantified using ImageQuant TL, version 8.1 (B). Values shown are means ± SD from three independent assays. C and D: Metabolic labeling of choline PL with radiolabeled choline. Cells were incubated with ¹⁴C-choline for the time indicated. After extraction, radioactive choline PL was analyzed by TLC (C). The amount of radiolabeled lipid in (C) was quantified and normalized by the amount of total cellular protein (D). E: The total radioactivity in the cells was measured using a liquid scintillation counter. Data are means ± SD from three independent culture dishes. ∗∗ and ∗∗∗ indicate P < 0.01 and P < 0.001 as compared with WT, respectively, determined using one-way ANOVA with Dunnett's post hoc test. F: Cell proliferation was assessed using Cell Counting Kit-8. The amount of the formazan dye generated by NADH dehydrogenases in cells is quantified by measuring absorbance at 450 nm.