Intellectual disability-associated gain-of-function mutations in CERT1 that encodes the ceramide transport protein CERT
Fig 5
The S135 mutations in CERT enhance its de novo SM synthesis activity.
(A) WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were analyzed by Western blotting with the indicated primary antibodies. (B) CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were fixed and analyzed by immunofluorescence microscopy. GM130 and VAP-A were immuno-stained with specific antibodies. The data are representative of two independent experiments. The areas enclosed by rectangles are enlarged (insets). Scale bars, 10 μm and 1 μm (inset). (C) WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were cultured with 250 ng/ml of lysenin for 1 hr. Next, cell viability was measured with an LDH cytotoxicity assay. The data comprise the mean ± SEM; n = 3 (***, p < 0.001). (D) The lipids in WT and CERT1 KO HCT116 cells stably expressing various mVenus-CERT constructs were metabolically labelled with L-[U-14C]serine. The labeled lipids were analyzed by TLC and visualized using an image analyzer. The data comprise the mean ± SEM; n = 3 (*, p < 0.05; ***, p < 0.001).