Journal: Journal of Cell Science
Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Figure Lengend Snippet: EMC loss does not activate a cholesterol homeostatic response. (A) Isolation of cytoplasmic (C) and nuclear (N) fractions from WT and ΔEMC6 (Δ6) cells by subcellular fractionation. Cells were treated with MBCD (1 mM), 25-hydroxycholesterol (25HC, 25 µM) or ethanol (control, Veh.) for 4 h. Western blots probing for the full-length (FL) and cleaved N-terminal fragment (N) of SREBP-2 are shown, along with Sec61β and histone H3 as controls for cytoplasmic and nuclear fractions, respectively. The asterisk (*) denotes a nonspecific band. (B) mRNA levels of HMGCR, SQS and SM as determined by quantitative real-time RT-PCR (qRT-PCR). Transcript levels present in untreated ΔEMC5 (Δ5) and Δ6 U2OS Flp-In™ T-Rex™ cells relative to WT are shown as means±s.d. ( n =6). (C) Western blots of whole-cell lysates derived from WT, Δ5 and Δ6 cells grown in normal medium were probed with the indicated antibodies, where Hsp70 served as a loading control. (D) Filipin III staining of WT and Δ6 cells maintained in DMEM plus 10% FCS to monitor intracellular cholesterol distribution. (E) mRNA levels of HMGCR, SM and SQS from WT and Δ6 cells treated with 1 mM MBCD for 0, 2 and 4 h, as determined by qRT-PCR relative to WT. Means±s.d. are shown ( n =3).
Article Snippet: Chemicals and compounds The following compounds were used: 25-hydroxycholesterol (25HC, Sigma-Aldrich, St. Louis, MO), DAPI (Sigma-Aldrich), aminooxybiotin (Biotium, Fremont, CA), aniline hydrochloride (Sigma-Aldrich), avasimibe (Sigma-Aldrich), cholesterol (Sigma-Aldrich), doxycycline (Sigma-Aldrich), filipin III (Santa Cruz Biotechnology, Dallas, TX), gel filtration markers kit (MWGF1000, Sigma-Aldrich), GGTI 298 trifluoroacetate salt hydrate (Sigma-Aldrich), Hygromycin B Gold (InvivoGen, San Diego, CA), IAA (iodoacetamide, Sigma-Aldrich), lipoprotein-depleted fetal bovine serum (LPDS, Kalen Biomedical, Germantown, MD), LMNG (lauryl maltose neopentyl glycol, Anatrace, Maumee, OH), methyl-β-cyclodextrin (MBCD, Sigma-Aldrich), MG132 (Merck Chemicals Ltd, Darmstadt, Germany), N-ethylmaleamide (NEM, Thermo Fisher Scientific), propidium iodide (Sigma-Aldrich), puromycin (Invivogen), SILAC Lys-8-Arg-10 kit (282986444, Silantes GmbH, Munich, Germany), sodium-meta-periodate (Sigma-Aldrich) and ZA (zaragozic acid A, Santa Cruz Biotechnology).
Techniques: Isolation, Fractionation, Western Blot, Quantitative RT-PCR, Derivative Assay, Staining