(A) Atom-transition map showing the isotope carbon-13 (¹³C) transfers from [1,2-¹³C₂]choline through the PC synthetic pathway. Open circles represent carbon-12 (¹²C); blue circles indicate ¹³C from [1,2-¹³C₂]choline. (B and C) Normalized peak areas of m+2 ¹³C-labeled metabolites from p53^+/+ and p53^–/– HepG2 cells cultured with choline-free medium and pulse-labeled with [1,2-¹³C₂]choline. (B) ¹³C-labeled choline, phosphocholine, and citicoline. (C) ¹³C-labeled PC. The isotopic labeling of each metabolite is denoted as m + n, where n is the number of ¹³C atoms. n = 3 samples per treatment. (D and F) Mice were fed a choline-deficient or normal diet for 4 weeks (n = 3 mice per group). Liver tissues were analyzed by quantitative reverse transcriptase PCR (RT-PCR) (D) and Western blot (F). (E and G) HepG2 cells cultured in choline-free medium or complete medium were analyzed by quantitative RT-PCR (E) and Western blot (G). (H) Immunohistochemistry of mouse liver tissues. Scale bar: 100 μm. (I) HepG2 cells were cultured with [1,2-¹³C₂]choline for 30 minutes, and relative PCYT1 activity was determined. (J) Liver tissue from p53^+/+ mice fed a normal or choline-free diet was lysed and immunoprecipitated with anti-p53 or IgG antibody. The bound DNA was amplified by quantitative RT-PCR. (K) Luciferase constructs containing WT or mutant response elements (REs) were transfected into HEK93T cells together with FLAG-p53 or vector control. Renilla vector pRL-CMV was used as a transfection internal control. Relative luciferase activity was calculated by division of FLAG-p53 samples over the FLAG vector control samples. Data are mean ± SD. Each experiment was carried out at least 3 times. P values were calculated by 2-tailed unpaired Student’s t test (B, C, and I–K) or 2-way ANOVA with Tukey’s multiple-comparison test (D and E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.