scd1 inhibitor delivery  (MedChemExpress)

Journal: eLife

Article Title: Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 ( Adgrf1 )

doi: 10.7554/eLife.85131

Figure Lengend Snippet: Primary hepatocytes were isolated from 8-week-old male C57BL/6J mice with STC. ( A ) Primary hepatocytes were infected with either adenoviral vector expressing Adgrf1 (ADV- Adgrf1 ) or control adenovirus expressing GFP (ADV- GFP ) 24 hr after plating, followed by transfection with ASO1- Adgrf1 , ASO2- Adgrf1, or ASO-NC for another 6 hr (n = 6). mRNA expression levels of Adgrf1 and Scd1 from different groups were assessed, as determined by RT-qPCR analysis. ( B ) Left panel: immunoblotting analysis for the expression level of Adgrf1 and Scd1 from different groups of primary hepatocytes. Right panel: quantification of protein expression levels of Adgrf1 and Scd1. Protein expression levels were normalized to the expression of β-tubulin. Each lane is a sample from a different plate. Right panel: quantification of protein expression levels of Adgrf1, Scd1, and β-tubulin. n = 3 per group. Protein expression levels were normalized to the expression of β-tubulin. The samples for GFP were set as 1 for fold-change calculation. ( C, D ) HEK293T cells were infected with pGL3- Scd1 promoter-luciferase plasmid and adenoviral vector expressing Adgrf1 (ADV- Adgrf1 ) or GFP (ADV- GFP ) for 48 hr and DHEA was added to the transfected cells at the concentration of 100 μM for 24 hr. Cell lysates were used for ( C ) luciferase assay or ( D ) RT-qPCR analysis (n = 3). Lysates from the cell co-transfection with pGL3- Scd1 promoter-luciferase plasmid and ADV- GFP without treatment of DHEA was set as 1 for fold-change calculation. ( E–G ) Primary hepatocytes were infected with either adenoviral vector expressing Adgrf1 (ADV- Adgrf1 ) or control ADV- GFP , followed by transfecting with scramble or sh Scd1 -1 or sh Scd1 -2 plasmids for another 72 hr. Intracellular lipids were extracted and ( E ) CHO, ( F ) TG, and ( G ) FFA were assessed (n = 3). STC, standard chow diet; i.v., intravenous injection; s.c., subcutaneous injection; ASO, antisense oligonucleotides. CHO, cholesterol; TG, triglyceride; FFA, free fatty acid. Data represented as mean ± SEM; repeated with three independent experiments; p-value analyzed by two-tailed Student’s t -test. *p<0.05, **p<0.01, ***p<0.001. Figure 6—source data 1. Scd1 expression is regulated by Adgrf1 in primary hepatocytes.

Article Snippet: For Scd1 inhibitor delivery, MK-8245 (MedChemExpress, NJ) was gavage at 10 mg/kg once a week ( ).

Techniques: Isolation, Infection, Plasmid Preparation, Expressing, Control, Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Concentration Assay, Cotransfection, Injection, Two Tailed Test

Journal: eLife

Article Title: Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 ( Adgrf1 )

doi: 10.7554/eLife.85131

Figure Lengend Snippet: Eight-week-old male C57BL/6J mice were infected with either 3 × 10 11 copies of rAAV encoding Adgrf1 (rAAV- Adgrf1 , i.v.) or control (rAAV- GFP , i.v.) and Scd1 inhibitor (MK8245, 10 mg/kg/week, p.o.) or inhibitor vehicle (inhibitor-Veh., p.o.) received HFD feeding. ( A ) Schematic illustration of viral treatments. ( B ) Hepatic mRNA expression levels of Adgrf1 from different groups of mice received rAAV and inhibitor fed with HFD, respectively, as determined by RT-qPCR analysis. ( C ) Left panel: immunoblotting analysis for the hepatic protein expression level of Adgrf1 and Scd1 from different groups of mice fed with HFD. Right panel: quantification of protein expression levels of Adgrf1 and Scd1. Protein expression levels were normalized to the expression of β-tubulin. Each lane is a sample from a different individual. ( D ) BW and ( E ) fasting blood glucose level were measured at different weeks upon rAAV and inhibitor injection. ( F ) The fasting blood insulin level and ( G ) HOMA-IR index were measured and calculated according to the formula [Fasting blood glucose (mmol/l)×Fasting blood insulin (mIU/l)]/22.5 for the HFD-fed rAAV- Adgrf1 or rAAV- GFP mice at the end of the experiment. ( H ) GTT (1 g/kg BW, left) and AUC (right) of serum glucose at the week of 10. ( I ) PTT (1 g/kg BW, left) and AUC (right) of serum glucose at week 11. ( J ) ITT (0.5 U/kg BW, left) and AUC (right) of serum glucose at week of 12. mRNA expression levels of the target genes were normalized to the expression of mouse Gapdh . rAAV-NC group was set as 1 for fold-change calculation. n = 8 per group. HFD, high-fat diet; i.v., intravenous injection; p.o., oral administration; ASO, antisense oligonucleotides; BW, body weight; GTT, glucose tolerance test; PTT, pyruvate tolerance test; ITT, insulin tolerance test; AUC, area under curve; NC, negative control; HOMA-IR, homeostasis model assessment-estimated insulin resistance. Data represented as mean ± SEM; repeated with three independent experiments; p-value analyzed by two-tailed Student’s t -test. *p<0.05, **p<0.01, ***p<0.001. Figure 7—source data 1. Inhibition of Scd1 alleviates the glucose impairment in mice with hepatic Adgrf1 overexpression.

Article Snippet: For Scd1 inhibitor delivery, MK-8245 (MedChemExpress, NJ) was gavage at 10 mg/kg once a week ( ).

Techniques: Infection, Control, Expressing, Quantitative RT-PCR, Western Blot, Injection, Negative Control, Two Tailed Test, Inhibition, Over Expression

Journal: eLife

Article Title: Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 ( Adgrf1 )

doi: 10.7554/eLife.85131

Figure Lengend Snippet: Eight-week-old male C57BL/6N mice were infected with either 3 × 10 11 copies of rAAV encoding Adgrf1 (rAAV- Adgrf1 , i.v.) or control (rAAV- GFP , i.v.) and administered with Scd1 inhibitor (MK8245, 10 mg/kg, p.o.) or inhibitor vehicle (inhibitor-Veh., p.o.) received HFD feeding. ( A ) Serum CHO, ( B ) serum TG, and ( C ) serum FFA levels were measured at the end of experiment. ( D ) Serum HDL and LDL, ( E ) AST and ALT level of each group of mice were measured at the end of the experiment. ( F ) The ratio of the liver weight against body weight was calculated after sacrificing the mice from four different groups. ( G ) Representative gross pictures of liver tissues (upper panels), representative images of H&E (middle panels) and Oil Red O (lower panels) staining of liver sections (200 µm). The percentage of lipid area according to H&E staining (right panel). ( H ) Hepatic CHO, ( I ) hepatic TG, and ( J ) hepatic FFA were normalized by the weight of liver samples used for lipid extraction. n = 8 per group. STC, standard chow diet; HFD, high-fat diet; i.v., intravenous injection; p.o., oral administration; CHO, cholesterol; TG, triglyceride; FFA, free fatty acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate transaminase; ALT, alanine transaminase; H&E, hematoxylin-eosin. Data represented as mean ± SEM; repeated with three independent experiments; p-value analyzed by two-tailed Student’s t- test. *p<0.05, **p<0.01, ***p<0.001. Figure 8—source data 1. Inhibition of hepatic Scd1 partially alleviates the severity of hepatic steatosis in Adgrf1 overexpression mice.

Article Snippet: For Scd1 inhibitor delivery, MK-8245 (MedChemExpress, NJ) was gavage at 10 mg/kg once a week ( ).

Techniques: Infection, Control, Staining, Extraction, Injection, Two Tailed Test, Inhibition, Over Expression

Journal: eLife

Article Title: Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 ( Adgrf1 )

doi: 10.7554/eLife.85131

Figure Lengend Snippet: NAFLD patients have higher hepatic expression of ADGRF1 accompanied with increased mRNA Scd1 expression. ( A ) Normalized Log 2 mRNA expression of ADGRF1 in lean people without NAFLD (n = 12), obese people without NAFLD (n = 17), or obese patients with NAFLD (n = 8) according to the GEO database (GEO; Profile # GDS4881/8126820). ( B ) Correlation between ADGRF1 and SCD1 in liver of human subjects based on the GEO database. ( C ) Representative images of liver tissues with H&E staining (upper panels) and immunohistochemical staining (IHC) of ADGRF1 (lower panels) from patients with different degree of NAFLD (200 µm). The percentage of ADGRF1 positive area according to H&E staining (right panel). The percentage of ADGRF1 -positive areas according to IHC staining (right panel); n = 3 per group. ( D ) Normalized Log2 mRNA expression of IL-1β in lean people without NAFLD (n = 12), obese people without NAFLD (n = 17), or obese patients with NAFLD (n = 8) according to the GEO database (GEO; Profile # GDS4881/8126820). ( E ) Hepatic mRNA expression levels of lL-1β , ( F ) Adgrf1, and ( G ) Scd1 in either STC-fed mice or HFD-fed mice treated with CCl4 or STZ as determined by RT-qPCR. Data represented as mean ± SEM. p-Value analyzed by two-tailed Student’s t -test. *p<0.05, **p<0.01, ***p<0.001. Figure 9—source data 1. Hepatic expression of ADGRF1 is upregulated in obese patients with hepatic steatosis when compared to those with normal liver morphology, which is positively associated with hepatic SCD1 expression level.

Article Snippet: For Scd1 inhibitor delivery, MK-8245 (MedChemExpress, NJ) was gavage at 10 mg/kg once a week ( ).

Techniques: Expressing, Staining, Immunohistochemical staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test