Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: Micro-RNA 124a (miR-124a) regulates expression of adipose triglyceride lipase (ATGL) and CGI-58 . ( A ) pre-miR-124a or control pre-miRNA was co-transfected with or without miR-124a inhibitor in OP9 adipocytes and mRNA- ( A ) or protein-expression ( B ) of Atgl was determined; ( C , D ) Cgi-58 (mRNA)- ( C ) or protein-expression ( D ) was analyzed after co-transfecting pre-miR-124a or control pre-miRNA with or without miR-124a inhibitor into OP9 adipocytes; ( E – G ) Lentiviral particles carrying either control pre-miRNA or miR-124a were transduced into primary mouse adipocytes and qRT-PCR was performed for Atgl (mRNA) ( E ) or Cgi-58 (mRNA) ( F ); Immunocytochemical analysis highlights the lipid bound “active pool” of ATGL. Arrows depict ATGL staining on lipid droplets; ( G ) Data are shown as average ± standard deviation and represent three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Luciferase reporter plasmid carrying wt or mutated Atgl or Cgi-58 3'UTR regions were purchased from Origene (Origene, MD, USA) ( E,F).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: miR-124a interacts with the 3'UTR of Atgl (mRNA) and Cgi-58 (mRNA). ( A ) Luciferase activity (relative units, normalized to β-gal activity for transfection normalization) measured after co-transfection of HeLa cells with either LUC-MIR-ATGL or LUC-SCR-ATGL with control pre-miRNA or pre-miR-124a and miR-124a inhibitor as indicated; ( B ) LUC-MIR-CGI or LUC-SCR-CGI was co-transfected into HeLa cells together with either miR-124a or control pre-miRNA and miR-124a inhibitor. β-gal containing plasmid was transfected for transfection normalization. Values are presented as relative luciferase units normalized to β-gal activity; ( C , D ) HeLa cells were co-transfected with YFP-ATGL-miR ( C ) or CFP-CGI-miR ( D ) and either pre-miR-124a or control pre-miRNA. Data are shown as average ± standard deviation and represent three independent experiments. ** p < 0.01, * p < 0.05.

Article Snippet: Luciferase reporter plasmid carrying wt or mutated Atgl or Cgi-58 3'UTR regions were purchased from Origene (Origene, MD, USA) ( E,F).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: miR-124a negatively regulates lipolysis ( A , B ) OP9 adipocytes were transfected Z787632 with either control pre-miRNA or pre-miR-124a. TG hydrolase activities were analyzed in the presence or in the absence of 76-0079 (HSL inhibitor) ( A ) or 500 ng recombinant CGI-58 protein ( B ); ( C , D ) free fatty acid ( C ) and glycerol ( D ) release was quantified in OP9 adipocytes after transfection with either pre-miR-124a or control pre-miRNA with or without miR-124a inhibitor; ( E ) Primary mouse adipocytes transduced with lentiviral particles carrying either control pre-miRNA or miR-124a were stained with BODIPY 493/503 fluorescence dye; White arrows point towards the differences in lipid droplet size ( F ) OP9 adipocytes were transfected with either pre-miR-124a or control pre-miRNA with or without miR-124a inhibitor and cellular triglyceride (TG) was quantified. Data are shown as average ± standard deviation and represent three independent experiments. ** p < 0.01, * p < 0.05.

Article Snippet: Luciferase reporter plasmid carrying wt or mutated Atgl or Cgi-58 3'UTR regions were purchased from Origene (Origene, MD, USA) ( E,F).

Techniques: Transfection, Recombinant, Transduction, Staining, Fluorescence, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: miR-124a expression negatively correlates to Atgl (mRNA) levels in various murine tissues. ( A , B ) Relative Atgl (mRNA) ( A ) and protein ( B ) expression measured in various murine tissues. Atgl (mRNA) level in adipose tissue arbitrarily set to 10; ( C ) Relative expression of mature miR-124a measured by qRT-PCR. Mature miR-124a level in brain arbitrarily set to 10; ( D ) Correlation between Atgl (mRNA) and miR-124a expression in various murine tissues; ( E ) Relative Atgl , Cgi-58 and miR-124a expression measured in murine perirenal as well as gonadal WAT; ( F ) Relative Atgl , Cgi-58 and miR-124a expression measured in murine perirenal WAT from ad libitum fed or, 12 h overnight fasted, mice; ( G ) Relative Atgl , Cgi-58 and miR-124a expression measured in murine liver from ad libitum fed or, 12 h overnight fasted or, 12 h overnight fasted and 2 h re-fed mice. Data are shown as average ± standard deviation and represent three independent experiments ( A – D ) or five independent experiments ( E – G ). *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Luciferase reporter plasmid carrying wt or mutated Atgl or Cgi-58 3'UTR regions were purchased from Origene (Origene, MD, USA) ( E,F).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: Micro-RNA 124a (miR-124a) regulates expression of adipose triglyceride lipase (ATGL) and CGI-58 . ( A ) pre-miR-124a or control pre-miRNA was co-transfected with or without miR-124a inhibitor in OP9 adipocytes and mRNA- ( A ) or protein-expression ( B ) of Atgl was determined; ( C , D ) Cgi-58 (mRNA)- ( C ) or protein-expression ( D ) was analyzed after co-transfecting pre-miR-124a or control pre-miRNA with or without miR-124a inhibitor into OP9 adipocytes; ( E – G ) Lentiviral particles carrying either control pre-miRNA or miR-124a were transduced into primary mouse adipocytes and qRT-PCR was performed for Atgl (mRNA) ( E ) or Cgi-58 (mRNA) ( F ); Immunocytochemical analysis highlights the lipid bound “active pool” of ATGL. Arrows depict ATGL staining on lipid droplets; ( G ) Data are shown as average ± standard deviation and represent three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Antibodies used: ATGL (2138, Cell Signaling, Danvers, MA, USA), CGI-58 (H00051099-M01, Abnova, Taipei City, Taiwan), β-actin (sc-47778, Santa Cruz, CA, USA).

Techniques: Expressing, Control, Transfection, Quantitative RT-PCR, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: miR-124a interacts with the 3'UTR of Atgl (mRNA) and Cgi-58 (mRNA). ( A ) Luciferase activity (relative units, normalized to β-gal activity for transfection normalization) measured after co-transfection of HeLa cells with either LUC-MIR-ATGL or LUC-SCR-ATGL with control pre-miRNA or pre-miR-124a and miR-124a inhibitor as indicated; ( B ) LUC-MIR-CGI or LUC-SCR-CGI was co-transfected into HeLa cells together with either miR-124a or control pre-miRNA and miR-124a inhibitor. β-gal containing plasmid was transfected for transfection normalization. Values are presented as relative luciferase units normalized to β-gal activity; ( C , D ) HeLa cells were co-transfected with YFP-ATGL-miR ( C ) or CFP-CGI-miR ( D ) and either pre-miR-124a or control pre-miRNA. Data are shown as average ± standard deviation and represent three independent experiments. ** p < 0.01, * p < 0.05.

Article Snippet: Antibodies used: ATGL (2138, Cell Signaling, Danvers, MA, USA), CGI-58 (H00051099-M01, Abnova, Taipei City, Taiwan), β-actin (sc-47778, Santa Cruz, CA, USA).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Control, Plasmid Preparation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: miR-124a negatively regulates lipolysis ( A , B ) OP9 adipocytes were transfected Z787632 with either control pre-miRNA or pre-miR-124a. TG hydrolase activities were analyzed in the presence or in the absence of 76-0079 (HSL inhibitor) ( A ) or 500 ng recombinant CGI-58 protein ( B ); ( C , D ) free fatty acid ( C ) and glycerol ( D ) release was quantified in OP9 adipocytes after transfection with either pre-miR-124a or control pre-miRNA with or without miR-124a inhibitor; ( E ) Primary mouse adipocytes transduced with lentiviral particles carrying either control pre-miRNA or miR-124a were stained with BODIPY 493/503 fluorescence dye; White arrows point towards the differences in lipid droplet size ( F ) OP9 adipocytes were transfected with either pre-miR-124a or control pre-miRNA with or without miR-124a inhibitor and cellular triglyceride (TG) was quantified. Data are shown as average ± standard deviation and represent three independent experiments. ** p < 0.01, * p < 0.05.

Article Snippet: Antibodies used: ATGL (2138, Cell Signaling, Danvers, MA, USA), CGI-58 (H00051099-M01, Abnova, Taipei City, Taiwan), β-actin (sc-47778, Santa Cruz, CA, USA).

Techniques: Transfection, Control, Recombinant, Transduction, Staining, Fluorescence, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

doi: 10.3390/ijms16048555

Figure Lengend Snippet: miR-124a expression negatively correlates to Atgl (mRNA) levels in various murine tissues. ( A , B ) Relative Atgl (mRNA) ( A ) and protein ( B ) expression measured in various murine tissues. Atgl (mRNA) level in adipose tissue arbitrarily set to 10; ( C ) Relative expression of mature miR-124a measured by qRT-PCR. Mature miR-124a level in brain arbitrarily set to 10; ( D ) Correlation between Atgl (mRNA) and miR-124a expression in various murine tissues; ( E ) Relative Atgl , Cgi-58 and miR-124a expression measured in murine perirenal as well as gonadal WAT; ( F ) Relative Atgl , Cgi-58 and miR-124a expression measured in murine perirenal WAT from ad libitum fed or, 12 h overnight fasted, mice; ( G ) Relative Atgl , Cgi-58 and miR-124a expression measured in murine liver from ad libitum fed or, 12 h overnight fasted or, 12 h overnight fasted and 2 h re-fed mice. Data are shown as average ± standard deviation and represent three independent experiments ( A – D ) or five independent experiments ( E – G ). *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Antibodies used: ATGL (2138, Cell Signaling, Danvers, MA, USA), CGI-58 (H00051099-M01, Abnova, Taipei City, Taiwan), β-actin (sc-47778, Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation