Fig 4. The CERT S135P mutant enhances de novo SM synthesis in HCT116 cells.

(A) WT (HCT116 WT) and CERT1 KO HCT116 cells stably expressing various HA-CERT constructs were analyzed by Western blotting. (B) WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were cultured with L-[U-¹⁴C]serine for 16 hr. Metabolically labeled lipids were analyzed by TLC. The data comprise the mean ± SEM; n = 4 (*, p < 0.05). (C) WT and CERT1 KO HCT116 cells stably expressing the indicated mVenus-CERT constructs were cultured with 250 ng/ml of lysenin for 1 hr. The cell viability was measured with a lactate dehydrogenase (LDH) cytotoxicity assay. The data comprise the mean ± SEM; n = 4 (*, p < 0.05; **, p < 0.01). (D) CERT1 KO HCT116 cells stably expressing the indicated mVenus-CERT constructs were analyzed by immunofluorescence microscopy. The data are representative of two independent experiments. Areas enclosed by rectangles are enlarged (insets). Scale bars, 10 μm and 1 μm (inset).