Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Protein Targeting of Phospholamban: A Common Role for an N-Terminal Di-Arginine Motif in ER Retention?

doi: 10.1371/journal.pone.0011496

Figure Lengend Snippet: ( A ) Subcellular fractionation of wild type Sigma 1R and a di-arg to di-glu mutant Sigma 1R construct. HEK cells were transiently transfected with tagged V5 Sigma 1R or tagged V5 di-arg to di-glu mutant Sigma 1R and cell lysates run on a continuous 20–60% sucrose gradient. Sigma1R was detected using the anti-V5 antibody. ( B ) IP performed using cell lysates from wild type V5 tagged Sigma 1R and V5 tagged di-arginine mutant Sigma 1R constructs transfected HEK cells. Proteins were immunoprecipitated using anti COP I antibody and then immunoblotted with anti V5 antibody (right panel); with equivalent loading of lysates (left panel). Arrows indicate Sigma 1R and mutant Sigma 1R. ( C ) Subcellular fractionation of wild type SR-β and di-arginine mutant SR-β construct. HEK cells were transiently transfected with V5 tagged SR-β or V5 tagged di-arginine mutant SR-β and cell lysates run on a continuous 20–60% sucrose gradient. SRB was detected using anti-V5 antibody. ( D ) IP performed using cell lysates from wild type V5 tagged SR-β and V5 tagged di-arginine mutant SR-β constructs transfected HEK cells and immunoprecipitated using anti COP I antibody and then immunoblotted with anti V5 antibody (right panel); with equivalent loading of lysates (left panel). Arrows indicate SR-β and mutant SR-β ( E ) Subcellular fractionation of wild type SOAT and di-arginine mutant SOAT construct. HEK cells were transiently transfected with tagged SOAT and di-arginine mutant SOAT and cell lysates run on a continuous 20–60% sucrose gradient and detected using anti-SOAT antibody ( F ) Tandem affinity purification of HEK cells transiently transfected with tagged SOAT and di-arginine mutant SOAT. Purification was carried out using streptavidin beads first and calmodulin beads second. Samples were then probed with antibodies to detect COP I, arrows indicate COP I (see for details).

Article Snippet: Due to cloning difficulties, SOAT cDNA (OriGene) was cloned into the Interplay Mammalian TAP vector, pCTAP (Stratagene) which contains a dual streptavidin binding peptide tag and a calmodulin binding peptide tag.

Techniques: Fractionation, Mutagenesis, Construct, Transfection, Immunoprecipitation, Affinity Purification, Purification

Journal: Endocrinology

Article Title: Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

doi: 10.1210/en.2015-1367

Figure Lengend Snippet: Figure 5. SOAT1 is expressed predominantly in the endo-membranes of adrenocortical cells and tissues and is inhibited my mitotane. A, Immunoblot demonstrating differential expression of SOAT isoenzymes in NCI-H295 cells and nonsteroidogenic cell lines. Whereas SOAT 1 is highly expressed in NCI-H295 cells, only low expression is observed in all other cell lines studied. B, SOAT2 is prevailing in HEK293 and IMR32 cells (15 g total protein extract in lanes 1–6 and 25 g in lanes 7–12). C, Mitotane inhibits activity of ER-resident enzyme Sterol-O-Acyl-Transferase SOAT with an IC50 of 21 M. D, Subcellular fractionation of NCI-H295 cells incubated with 4- [125I]iodomitotane shows high uptake of mitotane in endomembranes (characterized by EEA1 expression) and mitochondria associated membranes (characterized by MTC02 expression). The immunoblotting representation has been fitted to the uptake graph. SOAT1- immunhistochemistry of normal adrenal gland (E, 1adrenal cortex, 2 adrenal medulla), adrenal cortex (F, 1zona glomerulosa, 2zona fasciculate, 3zona reticularis) and cortisol producing (G), aldosterone producing (H) and endocrine inactive (I) adrenocortical adenomas.

Article Snippet: 1 106 HEK293 cells were grown on chamber slides and transiently transfected using Lipofectamine 2000 reagent (Life Technologies) according to the manufaturer’s instructions using plasmids encoding SOAT1 (Origene, RC205774), SOAT2 (Origene, RC221499) or empty vector, respectively.

Techniques: Western Blot, Quantitative Proteomics, Expressing, Activity Assay, Fractionation, Incubation

Journal: Endocrinology

Article Title: Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

doi: 10.1210/en.2015-1367

Figure Lengend Snippet: Figure 6. Schematic model of mitotane molecular mechanism in adrenocortical cells. Inhibition of SOAT1 leads to reduction of cholesterol esters (CE, green) and increase of free cholesterol (CHOL) and fatty acids (FA) that trigger ER-stress. IRE1-dependent XBP1-mRNA splicing and subsequent nuclear localization mediates transcription of unfolded protein response (UPR) genes. As ER-stress persists, increased expression of PERK and subsequent eIF2 phosphorylation induces increase in CHOP expression, which triggers proapoptotic BAX and represses antiapoptotic Bcl-2 expression thus inducing cell apoptosis. FA and CHOL accumulation repress SREBF expression leading to reduced transcription of sterol responsive genes and down-regulation of steroidogenesis. Bold arrows indicate experimental evidence for increased (red) and decreased (blue) mRNA and/or protein expression.

Article Snippet: 1 106 HEK293 cells were grown on chamber slides and transiently transfected using Lipofectamine 2000 reagent (Life Technologies) according to the manufaturer’s instructions using plasmids encoding SOAT1 (Origene, RC205774), SOAT2 (Origene, RC221499) or empty vector, respectively.

Techniques: Inhibition, Expressing, Phospho-proteomics

Journal: PLoS ONE

Article Title: Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells

doi: 10.1371/journal.pone.0187560

Figure Lengend Snippet: (A) The mRNA accumulations of SOAT1 during embryogenesis in YSM of Japanese quail. (B-I) EECs and hepatocytes were treated for 24 hours with different concentrations of a fatty acid, OA (oleic acid) or PA (palmitic acid); L(low) = 10 μM; H (high) = 100 μM, or glucose (3.3, 5.5 or 17.5 mM), or hormones (estrogen L = 1.8 nM, H = 18 nM; Dex L = 1 μm, H = 10 μm, glucagon L = 10 nM, H = 100 nM; IBMX L = 0.05 mM, H = 0.5 mM). SOAT1 mRNA accumulation was measured by Real-Time PCR and normalized to β-actin mRNA accumulation. Data were expressed as mean ± S.E.M. (n = 6) Statistical significance was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05).

Article Snippet: SOAT1 was detected with rabbit anti-mouse SOAT1 primary antibody (antibody diluted 1:500, orb100781, Biorbyt, Cambridgeshire, UK) followed by incubation with anti-rabbit IgG HRP-linked secondary antibody (1:5000, 7074P2, Cell Signaling, Danvers, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells

doi: 10.1371/journal.pone.0187560

Figure Lengend Snippet: (A-D) Effects of forskolin and dibutyryl-cyclic AMP (db-cAMP) on SOAT1 mRNA accumulations in EECs and hepatocytes. EECs and hepatocytes were treated with different concentrations of forskolin or db-cAMP for 24 hours. SOAT1 mRNA accumulation was measured and normalized to β-actin mRNA. (n = 4) (E-F) H89 inhibited IBMX-induced SOAT1 mRNA accumulations in EECs and hepatocytes. SOAT1 mRNA accumulation was increased by 0.5 mM IBMX treatment for 24 hours, whereas the PKA inhibitor, H89 (10 μM) abolished the IBMX-mediated increase of SOAT1 . (n = 6) (G-H) The IBMX treatment increased PKA activity in EECs and hepatocytes. The cells were treated with 0.5 mM IBMX with or without 10 μM H89 for 24 hours. The cell lysates were assayed for PKA activity using the PepTag assay. Typical gel patterns are represented (three independent experiments) for EECs and hepatocytes with statistical analysis of the densitometric analysis represented by the graphs (quantified by ImageQuant software). (I) Fluorescence examination of SOAT1 activity by applying NBD-cholesterol as the substrate. EECs were incubated with 10 μg/mL of NBD-cholesterol at 37°C for 30 mins or 1 hour. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. (J-K) The fluorescent intensity was quantified by ImageJ software. EECs were treated with or without db-cAMP for 24 hours. EECs were then incubated with 10 μg/mL of NBD-cholesterol for 30 min or 1 hour and examined by confocal microscopy. (J) Fluorescent intensity of total area. K: Fluorescent intensity per area of lipid droplets in EECs. (n = 4) Statistical significance was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05). (L) The SOAT1 protein levels after db-cAMP or IBMX treatments. EECs were treated with 0.5 or 1 mM db-cAMP or 0.5 mM IBMX for 48 hours and protein levels were examined by Western blotting. The β-actin was used as internal control. Statistical significance was determined by one-way analysis of variance. Dunnett's multiple comparisons test was used to evaluate differences between means. Data were expressed as means ± S.E.M. (n = 8) and different letters indicate the significant difference (P≤0.05). (M-N) The effects of H89 with or without db-cAMP and IBMX treatments on NBD-cholesterol uptake and storage. (M) EECs were pre-treated with or without H89 (10 μM) for 2 hours, and added db-cAMP (1 mM) or IBMX (0.5 mM) for 24 hours. Cells were then incubated with 10 μg/mL NBD-cholesterol for one hour, fixed with 4% paraformaldehyde and finally examined by confocal microscopy. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. Scale bar = 50 μm. (N) The quantitation of fluorescent intensity on NBD-cholesterol uptake and storage by ImageJ software in cells treated with H89 with or without IBMX or db-cAMP. Data were expressed as means ± S.E.M. (n = 4) Statistical significance among more than three different experimental groups was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Different letters indicate a significant difference (P≤0.05).

Article Snippet: SOAT1 was detected with rabbit anti-mouse SOAT1 primary antibody (antibody diluted 1:500, orb100781, Biorbyt, Cambridgeshire, UK) followed by incubation with anti-rabbit IgG HRP-linked secondary antibody (1:5000, 7074P2, Cell Signaling, Danvers, MA, USA).

Techniques: Activity Assay, Software, Fluorescence, Incubation, Staining, Confocal Microscopy, Control, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells

doi: 10.1371/journal.pone.0187560

Figure Lengend Snippet: The chicken primers for real-time PCR quantification.

Article Snippet: SOAT1 was detected with rabbit anti-mouse SOAT1 primary antibody (antibody diluted 1:500, orb100781, Biorbyt, Cambridgeshire, UK) followed by incubation with anti-rabbit IgG HRP-linked secondary antibody (1:5000, 7074P2, Cell Signaling, Danvers, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: PLoS ONE

Article Title: Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells

doi: 10.1371/journal.pone.0187560

Figure Lengend Snippet: (A) Effect of IBMX and the PKA inhibitor, H89 on the induction of SOAT1 promoter activity. Promoter activities were normalized for transfection efficiency using the renilla luciferase activity. (n = 5) (B) Effect of IBMX on the induction of serially deleted promoter regions of SOAT1 . The 293T cells were transfected with variable length constructs of the SOAT1 promoter and promoter activities were assayed after 24 hours of IBMX treatment. The pGL3 was a luciferase vector without SOAT1 promoter insertion, and was used as a negative control. (n = 6) (C) Diagram of wild type and mutated cAMP-responsive element on the SOAT1 promoter construct in a luciferase vector. (D) Effect of IBMX and H89 on the induction of promoter activity by the mutated CREB in the SOAT1 promoter. The 293T cells were transfected with normal or mutated SOAT1 promoter, and promoter activities were assayed after 0.5 mM IBMX and 10 μM H89 (PKA inhibitor) treatments for 24 hours. (n = 6) Data were expressed as means ± S.E.M. (n = three independent experiments) Statistical significance was determined by one-way analysis of variance. Tukey's test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05).

Article Snippet: SOAT1 was detected with rabbit anti-mouse SOAT1 primary antibody (antibody diluted 1:500, orb100781, Biorbyt, Cambridgeshire, UK) followed by incubation with anti-rabbit IgG HRP-linked secondary antibody (1:5000, 7074P2, Cell Signaling, Danvers, MA, USA).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Negative Control, Control

Journal: eLife

Article Title: A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection

doi: 10.7554/eLife.83534

Figure Lengend Snippet:

Article Snippet: Antibody , anti-ACAT1 (Rabbit Polyclonal) , Novus , CatNB400-141; RRID: AB_10001588 , WB (1:1,000).

Techniques: Transfection, Construct, Flow Cytometry, Recombinant, Sequencing, Bicinchoninic Acid Protein Assay, Microscopy, Protease Inhibitor, Modification, Saline, Methylation, Software, CRISPR, Imaging