Figure 2

MYC acted on lymphoma choline metabolism by regulating PCYT1A expression. (a) Schematic description of choline metabolism. PCYT1A, phosphate cytidylyltransferase 1 choline-α; PLA2G4C, phospholipase A; PC, phosphatidylcholine; LPC, lysophoaphatidylcholine. (b) HEK-293T cells were transfected with pCMV6-MYC plasmid or control plasmid (pCMV6-ct). The efficiency of transfection was confirmed by western blot (left panel). Expression of PCYT1A and PLA2G4C were assessed by quantitative real-time PCR (middle panel). HEK-293T cells were transfected with promoter-driven luciferase reporter of PCYT1A and PLA2G4C. Luciferase reporter activity was measured at 24 h after transfection (right panel). (c) Schematic diagram represented the regulatory region of PCYT1A promoter (upper panel). The activity of MYC binding to the PCYT1A promoter was measured by chromatin immunoprecipitation assay in DB cells either treated with 10058-F4 (40 μM) for 48 h or transfected with MYC siRNA. DNA–protein complexes from DB cells were precipitated with anti-MYC antibody and amplified with primers amplifying MYC binding sites on PCYT1A promoter using PCR and quantitative real-time PCR (lower panel). Antibody against RNA Polymerase II was referred as positive control. Nonspecific IgG was referred as negative control. (d) Schematic diagram represented the PCYT1A promoter reporters with or without mutation of MYC binding site (left panel). HEK-293T cells were transfected with mutated promoter-driven luciferase reporter of PCYT1A. Luciferase reporter activity was measured at 24 h after transfection (right panel). (e) Co-immunoprecipitation showed increased formation of RIP1/RIP3/MLKL complex in DB and Ramos cells, either treated with 10058-F4 (DB, 40 μM; Ramos, 30 μM) for 48 h or transfected with MYC siRNA. (f) Transmission electron microscopy showed typical necroptotic cells after 10058-F4 treatment or MYC siRNA transfection. Data in b, c and d were represented as mean±s.e.m. Assays in b, c and d were set up in triplicate.