pgl2-basic-luc  (Becton Dickinson)

Journal: Metabolism: clinical and experimental

Article Title: Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

doi: 10.1016/j.metabol.2019.01.014

Figure Lengend Snippet: Stamp2 promoter is activated by TNFα and p50. (A) 3T3-L1 cells with stable expression of either the −2kb-ST2-LUC or a BASIC-LUC reporters were differentiated into adipocytes, treated with TNFα (10 ng/ml) or vehicle (Ctrl) for 24 h and subjected to qRT-PCR analysis and (B) LUC assay. Values are presented as fold activity compared to vehicle. Results are from two independent experiments, n=9 for each sample. (C) HeLa cells were co-transfected with either 2xNFκB-LUC or −2kb-ST2-LUC (ST2-LUC), together with either AllStars (siAll) or NFKB1 (siNFKB1) siRNA. After 48 h, cells were harvested and LUC activity was determined. Values are presented as fold activity compared to control (siALL) from two independent experiments, n=6 for each sample. (D) qRT-PCR analysis of NFKB1 (p50) mRNA from the same cells used in C. Values are presented as fold activity compared to control (siALL). Results are from two independent experiments, n=6 for each sample. (E) HeLa cells were co-transfected with either 2xNFκB-LUC or −2kb-ST2-LUC (ST2-LUC), together with either a p50 expression plasmid or an empty control plasmid. After 48 h, cells were harvested and LUC activity was determined. Values are presented as fold activity compared to control (Ctrl) from two independent experiments, n=6 for each sample.

Article Snippet: To make stable reporter cells, linearized pGL2-BASIC-LUC or pGL2-BASIC-2kb-ST2-LUC was co-transfected with the linearized selection vector pPur (Clontech) in a 5:1 ratio using Lipofectamine 2000 (Invitrogen) as transfection agent.

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Transfection, Plasmid Preparation

Journal: Metabolism: clinical and experimental

Article Title: Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

doi: 10.1016/j.metabol.2019.01.014

Figure Lengend Snippet: Stamp2 promoter activity requires C/EBPα. (A) 3T3-L1 cells stably expressing the −2kb-ST2-LUC reporter were differentiated into adipocytes. They were then left untreated or incubated with Tg (300 nM), or Tu (2 ng/ml) for 24 h. Cells were then harvested and LUC activity was determined. Results are from two independent experiments, n=8 for each sample. (B) 3T3-L1 cells with stable expression of the −2kb-ST2-LUC reporter were transfected with either AllStar (siAll) or Cepba (siCEBPA) targeted siRNA, and differentiated towards adipocytes for 4 days. Cells were then harvested and LUC activities were determined. Values are presented as fold activity compared to control (siALL) from two independent experiments, n=6 for each sample. (C) qRT-PCR analysis of CEBPA mRNA from the same cells used in (B). (D) Overview of two predicted C/EBPα binding motifs (C/EBP1 and 2) in the mouse Stamp2 promoter. The nucleotides that are mutated are underlined. (E) HeLa cells were co-transfected with an expression vector for C/EBPα and either BASIC-LUC reporter plasmid (Ctrl), the wild-type −2kb-ST2-LUC, or a −2kb-ST2-LUC reporter construct mutated in C/EBP1 or C/EBP2 binding sites (MUT1 and MUT2, respectively). After 24 h, cells were harvested and LUC activities were determined. The data shown are from three independent experiments, n=9 for each sample.

Article Snippet: To make stable reporter cells, linearized pGL2-BASIC-LUC or pGL2-BASIC-2kb-ST2-LUC was co-transfected with the linearized selection vector pPur (Clontech) in a 5:1 ratio using Lipofectamine 2000 (Invitrogen) as transfection agent.

Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Transfection, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Construct

Journal: Metabolism: clinical and experimental

Article Title: Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

doi: 10.1016/j.metabol.2019.01.014

Figure Lengend Snippet: Stamp2 promoter is activated by TNFα and p50. (A) 3T3-L1 cells with stable expression of either the −2kb-ST2-LUC or a BASIC-LUC reporters were differentiated into adipocytes, treated with TNFα (10 ng/ml) or vehicle (Ctrl) for 24 h and subjected to qRT-PCR analysis and (B) LUC assay. Values are presented as fold activity compared to vehicle. Results are from two independent experiments, n=9 for each sample. (C) HeLa cells were co-transfected with either 2xNFκB-LUC or −2kb-ST2-LUC (ST2-LUC), together with either AllStars (siAll) or NFKB1 (siNFKB1) siRNA. After 48 h, cells were harvested and LUC activity was determined. Values are presented as fold activity compared to control (siALL) from two independent experiments, n=6 for each sample. (D) qRT-PCR analysis of NFKB1 (p50) mRNA from the same cells used in C. Values are presented as fold activity compared to control (siALL). Results are from two independent experiments, n=6 for each sample. (E) HeLa cells were co-transfected with either 2xNFκB-LUC or −2kb-ST2-LUC (ST2-LUC), together with either a p50 expression plasmid or an empty control plasmid. After 48 h, cells were harvested and LUC activity was determined. Values are presented as fold activity compared to control (Ctrl) from two independent experiments, n=6 for each sample.

Article Snippet: To make stable reporter cells, linearized pGL2-BASIC-LUC or pGL2-BASIC-2kb-ST2-LUC was co-transfected with the linearized selection vector pPur (Clontech) in a 5:1 ratio using Lipofectamine 2000 (Invitrogen) as transfection agent.

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Transfection, Plasmid Preparation

Journal: Metabolism: clinical and experimental

Article Title: Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

doi: 10.1016/j.metabol.2019.01.014

Figure Lengend Snippet: Stamp2 promoter activity requires C/EBPα. (A) 3T3-L1 cells stably expressing the −2kb-ST2-LUC reporter were differentiated into adipocytes. They were then left untreated or incubated with Tg (300 nM), or Tu (2 ng/ml) for 24 h. Cells were then harvested and LUC activity was determined. Results are from two independent experiments, n=8 for each sample. (B) 3T3-L1 cells with stable expression of the −2kb-ST2-LUC reporter were transfected with either AllStar (siAll) or Cepba (siCEBPA) targeted siRNA, and differentiated towards adipocytes for 4 days. Cells were then harvested and LUC activities were determined. Values are presented as fold activity compared to control (siALL) from two independent experiments, n=6 for each sample. (C) qRT-PCR analysis of CEBPA mRNA from the same cells used in (B). (D) Overview of two predicted C/EBPα binding motifs (C/EBP1 and 2) in the mouse Stamp2 promoter. The nucleotides that are mutated are underlined. (E) HeLa cells were co-transfected with an expression vector for C/EBPα and either BASIC-LUC reporter plasmid (Ctrl), the wild-type −2kb-ST2-LUC, or a −2kb-ST2-LUC reporter construct mutated in C/EBP1 or C/EBP2 binding sites (MUT1 and MUT2, respectively). After 24 h, cells were harvested and LUC activities were determined. The data shown are from three independent experiments, n=9 for each sample.

Article Snippet: To make stable reporter cells, linearized pGL2-BASIC-LUC or pGL2-BASIC-2kb-ST2-LUC was co-transfected with the linearized selection vector pPur (Clontech) in a 5:1 ratio using Lipofectamine 2000 (Invitrogen) as transfection agent.

Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Transfection, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Construct