Journal: Journal of Biological Chemistry

Article Title: AMP-activated protein kinase α1 promotes atherogenesis by increasing monocyte-to-macrophage differentiation

doi: 10.1074/jbc.m117.779447

Figure Lengend Snippet: Figure 5. FoxO3a regulates AMPKα1 dependent autophagy induction. A, Protein expression of Foxo3a in bone marrow derived macrophages in WT or AMPKα1-/- mice after 5 days of M-CSF treatment. #P<0.01 vs. WT. Data are representative of five independent experiments. B, Peritoneal macrophages were isolated 3 days after thioglycollate injection in WT or AMPKα1-/- mice. Protein expression of Foxo3a was detected by western blot. #P<0.05 vs. WT. Data are representative of three independent experiments. C, THP1 cells were transfected with AMPKα1 or control siRNA for 48 h. Protein expression of Foxo3a was detected by western blot. #P<0.01 vs. control siRNA. Data are representative of three independent experiments. D, THP1 cells were transfected AMPKα1 or control siRNA for 48 h and real-time RT-PCR was performed to quantify mRNA levels of FoxO3a. #P<0.01 vs. control. (n=6). E, HEK293T cells were co-transfected with pGL3-enhancer FoxO3a promoter vector in combination with pCMV-AMPKα1 plasmid or control plasmid. Renilla was used as an internal control. After 24 h of transfection, the cells were analyzed for Firefly luciferase and Renilla luciferase activity using the dual luciferase reporter assay. Data are expressed as the fold increase over mock-transfected cells. #P<0.01 vs. control. (n=6). F, THP1 cells were transfected with FoxO3a siRNA or control siRNA for 48 h and real-time RT-PCR was performed to quantify mRNA levels of LC3. #P<0.01 vs. control siRNA. (n=6). G, THP1 cells were transfected with FoxO3a siRNA for 48 h and then treated with or without 5 ng/ml bafilomycin A1 for 6h. Protein expression of LC3 was detected by Western blot. #P<0.01 vs. control siRNA, *P<0.01 vs. control siRNA with bafilomycin A1 treatment. Data are representative of three independent experiments. H, THP1 cells were transfected with FoxO3a siRNA or control siRNA for 48 h and ULK1 protein expressions were detected by western blotting. P<0.01 vs. control siRNA. Data are representative of three independent experiments. I, THP1 cells were transfected with FoxO3a or control siRNA for 48 h and real-time RT-PCR was performed to quantify mRNA levels of ULK1. #P<0.01 vs. control. (n=6).

Article Snippet: HEK293T cells were co-transfected with pGL3enhancer FoxO3a promoter vector (FHRE-Luc was a gift from Dr. Michael Greenberg (Addgene plasmid # 1789) in combination with the pCMVAMPKα1 plasmid (Origene, cat# RC218572) or empty plasmid.

Techniques: Expressing, Derivative Assay, Isolation, Injection, Western Blot, Transfection, Control, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

Journal: Biochemical and biophysical research communications

Article Title: Identification and Characterization of a Transcriptional Silencer Upstream of the Human BRCA2 Gene

doi: 10.1006/bbrc.1999.1652

Figure Lengend Snippet: Cloning of the human BRCA2 gene silencer. (A) Sketch showing the promoter and the silencer regions in the context of the upstream sequence of human BRCA2 gene. (B) Relative CAT activity in cell extracts obtained from MCF-10F cells transfected with different pCATbasic and pCATenhancer constructs. (C) Relative CAT activity in cell extracts obtained from MCF-10F cells transfected with different pCATcontrol constructs having the silencer sequence. Results are means ± SE (n = 6).

Article Snippet: Transformation of cells and reporter gene assay ( 4 – 6 ) The following plasmid vectors, procured from Promega (Madison, WI), were employed in our study: pCATbasic, pCATenhancer, pCATcontrol, pSV-b-galactosidase, pGL3-basic, pGL3-enhancer, pGL3-control and pRL-TK ( 5 ).

Techniques: Cloning, Sequencing, Activity Assay, Transfection, Construct

Journal: Biochemical and biophysical research communications

Article Title: Identification and Characterization of a Transcriptional Silencer Upstream of the Human BRCA2 Gene

doi: 10.1006/bbrc.1999.1652

Figure Lengend Snippet: Role of Alu-sequence in the silencer for the inhibition of promoter activities. (A) Mutational analysis of the silencer. The CAT activity was obtained in the extracts from MCF-10F cells transfected with different pCATcontrol constructs having the silencer sequence deletion mutant fragments. Alu sequences are shown by solid bars. Results are means ± SE (n = 6). (B) Nucleotide sequence of the 221 bp silencer region. The Alu-repeats are underlined. The consensus elements known to bind to Alu-binding protein are bold-faced. (C) Pustell DNA matrix analysis of the 221-bp DNA (with itself). The presence of direct repeat sequence in this DNA is indicated by two parallel lines above and below the identity diagonal.

Article Snippet: Transformation of cells and reporter gene assay ( 4 – 6 ) The following plasmid vectors, procured from Promega (Madison, WI), were employed in our study: pCATbasic, pCATenhancer, pCATcontrol, pSV-b-galactosidase, pGL3-basic, pGL3-enhancer, pGL3-control and pRL-TK ( 5 ).

Techniques: Sequencing, Inhibition, Activity Assay, Transfection, Construct, Mutagenesis, Binding Assay

Journal: Biochemical and biophysical research communications

Article Title: Identification and Characterization of a Transcriptional Silencer Upstream of the Human BRCA2 Gene

doi: 10.1006/bbrc.1999.1652

Figure Lengend Snippet: Cloning of the human BRCA2 gene silencer. (A) Sketch showing the promoter and the silencer regions in the context of the upstream sequence of human BRCA2 gene. (B) Relative CAT activity in cell extracts obtained from MCF-10F cells transfected with different pCATbasic and pCATenhancer constructs. (C) Relative CAT activity in cell extracts obtained from MCF-10F cells transfected with different pCATcontrol constructs having the silencer sequence. Results are means ± SE (n = 6).

Article Snippet: Transformation of cells and reporter gene assay ( 4 – 6 ) The following plasmid vectors, procured from Promega (Madison, WI), were employed in our study: pCATbasic, pCATenhancer, pCATcontrol, pSV-b-galactosidase, pGL3-basic, pGL3-enhancer, pGL3-control and pRL-TK ( 5 ).

Techniques: Cloning, Sequencing, Activity Assay, Transfection, Construct