Fig 2: SOAT1 loss from EMC-deficient cells and attenuated cholesteryl ester formation. (A) Heat map representing the fold change of 117 lipid species in EMC5 (?5 #5-4) and EMC6 (?6 #3-9) deletion mutants relative to WT as identified by targeted metabolomic analysis (n=4). Lipid species are arranged according to major structural class. The 14 lipid species significantly altered in both ?5 and ?6 cells (P=0.05) are indicated (grey circles, black star). NL, neutral lipids; FA, fatty acids; AC, acyl carnitine; NAE, N-acylethanolamines; ST, sterols; PL, phospholipids; LPL, lysophospholipids; SL, sphingolipids; NEL, neutral ether lipids; PLE, phospholipid ethers; LPLE, lysophospholipid ethers. (B) Quantification of free cholesterol and cholesteryl esters in ?5 and ?6 cells relative to WT (dashed line). Means±s.e.m. are shown (n=4). ***P=0.001, ****P=0.0001 (Student's t-test). (C,D) Western blots of whole-cell lysates (WCL) for ?5 cells with or without the EMC5 expression vector (C) and ?6 cells with or without the EMC6 expression vector (D) probed for SOAT1 and indicated EMC subunits. (E) Schematic representation of the dual reporter construct used in F–G. mRNA encoding GFP separated by a P2A sequence from RFP and FLAG-tagged SOAT1. Translation results in ribosome skipping and the generation of GFP and RFP-3xFLAG-SOAT1 at equimolar ratios. Differences in stability between both gene products gives rise to altered RFP:GFP ratios, serving as a sensitive readout for protein stability. (F) Fluorocytometric RFP:GFP ratio in WT and ?5 cells with or without the EMC5 expression vector and transiently expressing GFP-P2A-RFP-3xFLAG-SOAT1. At 24 h post transfection, cells were treated with MG132 (5 µg/ml) or DMSO (vehicle control) for 8 h and analysed by flow cytometry. EV, empty vector control. (G) Quantification of three independent experiments as performed in F. Means±s.d. are shown (n=3). ****P=0.0001 (Student's t-test). (H) WT and ?6 cells were exposed to Chol:MBCD (25, 37.5 and 50 µM, 20 h) with or without 10 µM avasimibe (AVA, 20 h) and visualised by staining with Crystal Violet.