pgl3-promoter empty vector  (Promega)

Journal: Frontiers in Physiology

Article Title: GATA Binding Protein 3 Is a Direct Target of Kruppel-Like Transcription Factor 7 and Inhibits Chicken Adipogenesis

doi: 10.3389/fphys.2020.00610

Figure Lengend Snippet: KLF7 directly regulates GATA3 transcription. (A) Western blot identification of KLF7 expression vector pCMV-Myc- KLF7 . M1, the cell lysates from the immortalized chicken preadipocyte cell line (ICP2) transfected with pCMV-Myc vector; M2, the cell lysates from the preadipocytes transfected with pCMV-Myc- KLF7 . The western blot analysis was repeated in three independent experiments. (B) The effect of KLF7 overexpression on the endogenous gene expression of GATA3 in ICP2 cells. ICP2 cells were transfected with either pCMV-Myc- KLF7 (KLF7) or pCMV-Myc (EV). Forty-eight hours after transfection, total RNA was isolated and the mRNA expression levels of GATA3 were measured using qRT-PCR. TBP was used as an internal control. Values represent mean ± SD from three independent experiments with three replicates in each. Asterisks indicate significant difference (Student’s t -test), P < 0.01( ∗∗ ). (C) Schematic diagram of GATA3 reporter constructs. A ∼950-bp genomic DNA fragment spanning −5285 to −4336 bp upstream of the translation initiation codon ATG of GATA3 was amplified by PCR and cloned into pGL3-Promoter vector. In the pGL3- GATA3 -DM construct, the CGCCGGG core sequence is deleted and shown as a dashed line. (D) Reporter gene analysis of the upstream region (−5285/−4336) of GATA3 gene in ICP2 and DF-1 cells. The cells were transfected with either pGL3- GATA3 or pGL3-Promoter (empty vector), and pRL- TK Renilla luciferase vector (100:1). After 48 h of cotransfection, relative luciferase activity was measured. The relative activity was expressed as the ratio of firefly to Renilla luciferase activity. Values represent mean ± SD from three independent experiments with three replicates in each. Asterisks indicate significant difference (Student’s t -test), P < 0.01( ∗∗ ). (E,F) The effect of KLF7 overexpression on the reporter activity of the wild-type reporter pGL3- GATA3 and mutation reporter pGL3- GATA3 -DM in ICP2 (E) and DF-1 cells (F) . The cells were cotransfected with pGL3- GATA3 or pGL3- GATA3 -DM and pCMV-Myc- KLF7 (KLF7) or pCMV-Myc (EV), and pRL- TK Renilla luciferase vector (50:50:1). After 48 h of cotransfection, relative luciferase activity was measured. The relative activity was expressed as the ratio of firefly to renilla luciferase activity. Values represent mean ± SD from three independent experiments with three replicates in each. Asterisks indicate significant difference (student’s t- t est) P < 0.01 ( ∗∗ ), P < 0.05 ( ∗ ). (G) ChIP assay for KLF7 binding to the GATA3 upstream region (−4561/−4439). ChIP assays were performed using the c-Myc tag antibody and purified mouse IgG, which was used as a negative control (Mock). Immunoprecipitated DNA samples were purified and analyzed by qPCR using two specific pairs of primers. One pair of primers for the GATA3 upstream region (−4561/−4439), and the other pair of primers for the coding genomic region (+39/+149) of GATA3 , which was used as a negative control (None). Non-immunoprecipitated DNA (1%) was used as input control. Data were presented as fold enrichment over the input control. Values represent mean ± SD from three independent experiments with three replicates in each. Asterisks indicate significant difference (student’s t- t est), P < 0.01 ( ∗∗ ).

Article Snippet: For analysis of the reporter gene activity of the -5285 to -4336 upstream region of chicken GATA3 gene, cells were cotransfected with pGL3- GATA3 or pGL3-Promoter (empty vector), and pRL- TK Renilla luciferase vector (Promega; Ratio 100:1).

Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Over Expression, Isolation, Quantitative RT-PCR, Construct, Amplification, Clone Assay, Sequencing, Luciferase, Cotransfection, Activity Assay, Mutagenesis, Binding Assay, Purification, Negative Control, Immunoprecipitation

Journal: Immunity, Inflammation and Disease

Article Title: Corynebacterium tuberculostearicum , a human skin colonizer, induces the canonical nuclear factor‐κB inflammatory signaling pathway in human skin cells

doi: 10.1002/iid3.284

Figure Lengend Snippet: Corynebacterium tuberculostearicum ( C. t .) induces the expression of pTNF3′NF‐κB luciferase construct in HEKs and SCC cells, and the luciferase activity is attenuated by PS‐1145. HEKs (A) or SCC cells (B), naïve or transfected with pGL3 promoter empty vector (E.V.) or with pTNF3′NF‐κB plasmid were treated for 1 hour with PS‐1145 (10 μM) or vehicle control (DMSO) before infection with C. t . or treatment with TNF. After 6 hours, luciferase activities were measured and collected data, N = 5, was analyzed with the GraphPad Prism 6 software. A two‐way analysis of variance was performed with Tukey's correction for multiple comparisons. The Student t test was used to determine the significant difference between two treatment groups, * P < .05; ** P < .01; *** P < .001. Data are expressed as relative luciferase activity and depicted as box‐and‐whiskers plots showing the median, the 25th, and 75th quartiles, as well as a minimum to maximum values. DMSO, dimethyl sulfoxide; HEK, human epidermal keratinocyte; NF‐κB, nuclear factor‐κB; SCC, squamous carcinoma cell; TNF, tumor necrosis factor

Article Snippet: A complex of 1 μL Lipofectamine 2000 (catalog no. 11668‐027; Invitrogen) and 400 ng DNA of pTNF3′NF‐κB plasmid or PGL3 promoter empty vector (E.V., catalog no. E1761; Promega) diluted in 50 μL Opti‐MEM (catalog no. 31985; Gibco) was added to each well and incubated for 48 hours.

Techniques: Expressing, Luciferase, Construct, Activity Assay, Transfection, Plasmid Preparation, Infection, Software