Journal: Theranostics

Article Title: Therapeutic inhibition of miR-802 protects against obesity through AMPK-mediated regulation of hepatic lipid metabolism

doi: 10.7150/thno.49354

Figure Lengend Snippet: The influence of miR-802 expression on lipid metabolism in murine hepatocytes is AMPK dependent. ( A ) qRT-PCR analysis of gene expression related to lipo-metabolism in FL83B cells with miR-802 mimic or inhibitor treatment. ( B, C ) qRT-PCR analysis of Prkaa1 and Prkaa2 expression in FL83B cells upon transfection with miR-802 mimic or inhibitor. ( D, E ) Western blot analysis of protein levels of Prkab1, Prkaa1, Prkaa2 upon transfection with miR-802 mimic or inhibitor in FL83B cells. ( F ) Western blot analysis of phosphorylated AMPK and ACC in FL83B cells after stimulating with A769662, miR-802 mimic or A769662 + miR-802 mimic. ( G ) Western blot analysis of phosphorylated AMPK and ACC in FL83B cells after stimulating with miR-802 inhibitor, compound C or miR-802 inhibitor + compound C. ( H ) ACC activity in FL83B cells after treating with miR-802 mimic or inhibitor. Data are expressed as the mean ± s.e.m of 3 independent experiments with similar results. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The FL83B murine hepatocyte cell line derived in 1969 by Charity Waymouth at the Jackson Laboratory was kindly provided by the Liver Transplantation Center of Jiangsu Province Hospital.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Transfection, Western Blot, Activity Assay

Journal: Theranostics

Article Title: Therapeutic inhibition of miR-802 protects against obesity through AMPK-mediated regulation of hepatic lipid metabolism

doi: 10.7150/thno.49354

Figure Lengend Snippet: miR-802 regulates the expression of Prkab1 . ( A ) qRT-PCR quantification of Prkab1 expression in livers of uninfected and infected C57BL/6 mice fed with ND and HFD. (B) Representative figures of Prkab1/Albumin double immunostaining on liver sections of four groups of mice. ( C ) Relative intensity data of Prkab1/Albumin double immunostaining cells (5 random liver fields in each mouse, n = 6 mice per group). ( D ) Conservation of miR-802 target regions in the 3'UTR of Prkab1 . ( E ) Luciferase report assays for 293T cells transfected with pMIR-Report Luciferase vectors carrying wild type (WT) or mutated (MuT) 3'UTR of Prkab1 in the presence of miR-802 mimic or NC-mimic. ( F ) qRT-PCR quantification of miR-802 and Prkab1 expression in FL83B cells and MPHs treated with miR-802 mimic. ( G ) qRT-PCR quantification of miR-802 and Prkab1 expression in FL83B cells and MPHs when using miR-802 inhibitor. Data are expressed as the mean ± s.e.m of 3 (n = 3 in E, F, G) or 2 (n = 6 in A, B, C) repeated experiments. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The FL83B murine hepatocyte cell line derived in 1969 by Charity Waymouth at the Jackson Laboratory was kindly provided by the Liver Transplantation Center of Jiangsu Province Hospital.

Techniques: Expressing, Quantitative RT-PCR, Infection, Double Immunostaining, Luciferase, Transfection

Journal: Theranostics

Article Title: Therapeutic inhibition of miR-802 protects against obesity through AMPK-mediated regulation of hepatic lipid metabolism

doi: 10.7150/thno.49354

Figure Lengend Snippet: Prkab1 activated AMPK to promote the oxidation and suppress the synthesis of fatty acid. ( A-C ) qRT-PCR analysis of Prkab1 , Prkaa1 and Prkaa2 expression in FL83B cells upon transfection with si- Prkab1 or OE- Prkab1 . ( D, E ) Western blot analysis of protein levels of Prkab1, Prkaa1 and Prkaa2 in FL83B cells after transfecting with si- Prkab1 or OE- Prkab1 . ( F ) Western blot analysis of phosphorylated AMPK and ACC in FL83B cells with OE- Prkab1 , compound C or OE- Prkab1 + compound C treatment. ( G ) Western blot analysis of phosphorylated AMPK and ACC in FL83B cells with A769662, si- Prkab1 or A769662 + si- Prkab1 treatment. ( H ) qRT-PCR analysis of lipogenesis-related genes expression in FL83B cells when transfected with si- Prkab1 or OE- Prkab1 . ( I ) ACC activity assay of FL83B cells upon treatment of si- Prkab1 or OE- Prkab1. Error bars represented mean ± s.e.m of 3 independent repeat experiments. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The FL83B murine hepatocyte cell line derived in 1969 by Charity Waymouth at the Jackson Laboratory was kindly provided by the Liver Transplantation Center of Jiangsu Province Hospital.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Activity Assay

Journal: Theranostics

Article Title: Therapeutic inhibition of miR-802 protects against obesity through AMPK-mediated regulation of hepatic lipid metabolism

doi: 10.7150/thno.49354

Figure Lengend Snippet: Sjp40 inhibits miR-802 expression through suppressing CD36 and NF-κB signaling. ( A ) After treatment with Sjp40 (10 µg/ml) for 15 min, immunoblot analysis of CD36 (green), Sjp40 (red) and DAPI (blue) expression in FL83B cells. ( B ) Pierce pull-down polyhistidine assay was employed for identifying the interaction between Sjp40 and CD36. ( C ) qRT-PCR analysis of Prkab1 and miR-802 expression in palmitate and oleic acid-stimulated FL83B cells upon treatment with Sjp40 for 24 h. ( D ) qRT-PCR quantification of miR-802 and Prkab1 in the livers. ( E ) Western blot analysis of palmitate and oleic acid-stimulated AMPK-ACC pathway in FL83B cells in absence or presence of Sjp40. ( F ) Western blot analysis of palmitate and oleic acid-stimulated CD36 protein levels in FL83B cells in absence or presence of Sjp40. Data are expressed as the mean ± s.e.m. for each group, and are representative of one typical experiment out of three. ( G ) Levels CD36 in the livers of four groups of mice. Data are expressed as the mean ± s.e.m. of 6 mice for each group in one representative experiment. All experiments were repeated twice. ( H ) Western blot analysis of p-AMPK and p-ACC levels in CD36 -/- primary hepatocytes in absence or presence of Sjp40. ( I ) Western blot analysis of LKB1 levels in FL83B cells in absence or presence of Sjp40. ( J ) qRT-PCR analysis of miR-802 in FL83B cells upon overexpression of NF-κB. ( K, L ) Luciferase activity of miR-802 promotor. 293T cells transfected with reporter constructs that contained the miR-802 promoter were treated with pNF-κB. After 48 h, luciferase activity was analyzed and plotted. Error bars represented mean ± s.e.m of 3 independent repeat experiments. ( M ) Protein Levels of p65 and p-p65 in palmitate and oleic acid-stimulated FL83B cells after stimulating with Sjp40. ( N ) Protein levels p65 and p-p65 in the livers of four groups of mice. Data are expressed as the mean ± s.e.m. for each group, and are representative of one typical experiment out of three. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The FL83B murine hepatocyte cell line derived in 1969 by Charity Waymouth at the Jackson Laboratory was kindly provided by the Liver Transplantation Center of Jiangsu Province Hospital.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Luciferase, Activity Assay, Transfection, Construct