Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: SCP2 deficiency significantly attenuates diet-induced atherosclerosis. At 10 weeks of age, LDLR−/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. Aortae were dissected and prepared for en face analyses. The total area as well as the area occupied by the lesions was quantified using Axiovision software and expressed as percent lesion area. A, representative images. B, percent area occupied by the lesions in the aortic arch. C, percent area occupied by the lesions in the entire aorta. *, p < 0.05; individual p values are included in the text.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: SCP2 deficiency reduces plaque size as well as plaque necrosis in the aortic root. Paraffin-embedded aortic root sections (5 μm) were stained with H&E or Masson's trichrome, imaged, and analyzed by Axiovision software. A, representative H&E stained images of the aortic root of the indicated genotypes/sex. Scale bar = 100 μm. B, the total aortic root and area occupied by the lesions was quantified, and data (mean ± S.D., n = 6) are presented as percent lesion area. C, representative trichrome-stained images of the indicated genotypes/sex. Scale bar = 50 μm. D, the total and necrotic areas were quantified for all three aortic valve leaflets, and data (mean ± S.D., n = 12 leaflets) are presented as percent necrotic area. *, p < 0.05.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Staining, Software

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: SCP2 deficiency significantly reduces plasma cholesterol and triglyceride levels. At 10 weeks of age, LDLR−/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. After an overnight fast, mice were euthanized, and fasting plasma was collected. A, levels of total plasma cholesterol for indicated genotype and sexes. B, percent of total plasma cholesterol associated with the non-HDL or HDL fraction. C, percent of total plasma cholesterol associated with the non-HDL or HDL fraction in both genotypes normalized to total plasma cholesterol in LDLR−/− mice of the respective sex. D, linear regression analyses of total lesion area and plasma cholesterol; the observed coefficient of correlation (R) as significance of correlation (p value) is indicated. E, total plasma triglyceride for the indicated genotype and sexes. F, linear regression analyses of total lesion area and plasma triglyceride levels; the observed coefficients of correlation (R) as significance of correlation (p value) is indicated.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: SCP2 deficiency significantly reduces intestinal cholesterol absorption, and SCP2 knockdown in intestinal epithelial cells reduces cholesterol uptake. A, C57BL/6 (WT) or SCP2−/− mice were injected intravenously with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg body weight) and gavaged with [3H]cholesterol in olive oil (2 μCi in 200 μl) after 5 min. Intestinal cholesterol absorption was assessed by monitoring the radiolabel associated with plasma collected at the time of euthanasia. Data are presented as plasma DPM per microliter of plasma for the indicated genotype and sex. B, the entire length of the intestine, from the base of the stomach to the tip of the cecum, was divided into four equal parts (P1 to P4), and total RNA was isolated. The mRNA levels of indicated genes were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. C, total protein extracts from ileum segments P1 to P4 of WT and SCP2−/− mice as well as the colon (C) were analyzed by Western blotting for expression of SCP2; β-actin was used as the loading control. Human intestinal epithelial cells (HT-29) were transfected with scrambled or SCP2-specific siRNA as described under “Experimental procedures.” Total protein or RNA was extracted and used to determine the levels of SCP2. D, top, a representative Western blot. Bottom, levels of SCP2 mRNA quantified by quantitative PCR and SCP2 protein by densitometric analyses of Western blots. Data are presented as percent scrambled siRNA-transfected controls for the indicated SCP2 siRNA concentrations used. E, HT-29 cells transfected with scrambled siRNA or 53.3 nm SCP2-specific siRNA were incubated with [3H]-cholesterol, and total cellular uptake was monitored as described under “Experimental procedures.” Data (mean ± S.D., n = 6) are presented as DPM normalized to total cellular protein. *, p < 0.05.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Injection, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: SCP2 deficiency significantly reduces lipid secretion from liver and isolated hepatocytes. A, C57BL/6 (WT) or SCP2−/− mice were injected with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg of body weight), and blood samples were drawn at 0 and 3 h. Triglyceride secretion rates for indicated genotypes and sexes are presented. B and C, primary hepatocytes were prepared from C57BL/6 (WT) or SCP2−/− mice. Following incubation with [3H]oleic acid, radiolabel associated with secreted triglycerides (TG, B) or cholesteryl esters (C) was determined as described under “Experimental procedures” and normalized to cellular protein. Data are presented as DPM associated with the triglyceride or cholesteryl ester fraction in the total lipids extracted from the medium per milligram of total protein.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Isolation, Injection, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: SCP2 deficiency leads to reduced lipid accumulation in the liver without a change in the expression of lipogenic genes. A, liver tissue harvested from WD-fed LDLR−/− or LS mice were paraffin-embedded, and 5-μm sections were stained with H&E. Images were acquired using a Zeiss inverted microscope fitted with a digital camera. Scale bar = 50 μm. B, SCP2 mRNA levels in total liver RNA were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. Relative expression (mean ± S.D., n = 6) is shown. C, hepatic mRNA levels of the indicated genes were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. Relative expression (mean ± S.D., n = 6) is shown.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Expressing, Staining, Inverted Microscopy, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: Effects of SCP2 deficiency on cholesterol accumulation in and cholesterol efflux from macrophages. Thioglycolate-elicited macrophages were isolated from C57BL/6 (WT) and SCP2−/− mice and incubated with AcLDL (25 μg/ml) for 48 h. A and B, following two washes with PBS, cells were either fixed and stained with Oil Red O (A) or used for total lipid extraction and cholesterol mass measurement (B). Total cholesterol mass was normalized to total cellular protein, and data (mean ± S.D., n = 6) are presented as nanomoles per milligram of protein. C, total protein extracts of macrophages were subjected to Western blot analyses to assess SR-A expression; β-actin was used as a loading control. D, for measurement of cholesterol efflux, cells were loaded with AcLDL and labeled with [3H]-cholesterol for 48 h. Following a 24-h equilibration, cholesterol efflux to 10% FBS in the growth medium was monitored over time. Data (mean ± S.D., n = 6) are presented as percent efflux. *, p < 0.05.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: Isolation, Incubation, Staining, Mass Measurement, Western Blot, Expressing, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

doi: 10.1074/jbc.RA118.002290

Figure Lengend Snippet: Effects of SCP2 deficiency on biliary bile acid and cholesterol secretion. A and B, gall bladder bile was collected at the time of euthanasia, and the total volume was noted. Biliary bile acids (BA), cholesterol, and phospholipids (PL) were estimated as described under “Experimental procedures.” Data are presented as total bile acids (nanomoles) or FC (micrograms) in the bile normalized to total phospholipids (micrograms) in A and B, respectively.

Article Snippet: Quantitative RT-PCR was used to measure mRNA levels using a TaqMan assay (Hs00920780_m1), and the protein levels were measured by Western blot analyses using primary antibody 23006-1-AP (Proteintech Group), which recognizes both SCP2 and SCPx.

Techniques: