luciferase reported vector, pgl 3 -basic  (Promega)

Journal: PLoS ONE

Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts

doi: 10.1371/journal.pone.0066892

Figure Lengend Snippet: The (−2436/+149) region of the PAK3 promoter, containing several putative cJun/AP- binding sites, was cloned into the pGL 3 -Basic reporter vector. A: Luciferase promoter reporter assays performed in control cells, Rat1a-GFP, and Rat1a-J4 cells transiently transfected with empty vector, pGL 3 -Basic, and PAK3 promoter construct containing vector, pGL 3 -Basic-pPAK3 (−2436/+149). Cells were grown in the absence and presence of doxycycline. B: Luciferase promoter reporter assays performed in the parental rat fibroblast cell line, Rat1a, transfected with either pCMV-cJun or empty pCMV vector with the PAK3 promoter construct containing vector, pGL 3 -Basic-pPAK3 (−2436/+149). A plasmid containing four AP-1 binding sites, 4 X AP-1-Luc, was used as a control showing cJun activation. C: Luciferase reporter promoter assays for deletion constructs of the PAK3 promoter region (−2436/+149), (−2329/+149), (−684/+149) and (−179/+149) as well as a (−179/+149) with a mutated cJun/AP-1 binding site at position (+52/+60), in the presence and absence of doxycycline-induced cJun/AP-1 expression. D: Luciferase reporter assays, using the (−179/+149) PAK3 promoter construct, showing the effect of transient cJun inhibition on PAK3 promoter activity in the absence and presence of doxycycline induced cJun/AP-1 over-expression. Results show the mean±S.E. of experiments performed in triplicate and repeated at least three times. (*p≤0.05 and **p≤0.01).

Article Snippet: PCR using high fidelity Expand Plus DNA Polymerase (Roche) was performed on rat genomic DNA, the products were sub-cloned into the pGEM-T Easy vector (Promega) and excised for cloning into the luciferase reported vector, pGL 3 -Basic (Promega) using Mlu1 and Xho1 restriction enzymes.

Techniques: Binding Assay, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Construct, Activation Assay, Expressing, Inhibition, Activity Assay, Over Expression

Journal: Oncogene

Article Title: PTTG Acts As a STAT3 Target Gene for Colorectal Cancer Cell Growth and Motility

doi: 10.1038/onc.2013.16

Figure Lengend Snippet: STAT3 binds the PTTG promoter, activates PTTG transcription and induces PTTG expression. (A) Schematic representation of potential STAT binding motifs on the human PTTG promoter, and five primers designed for chromatin immunoprecipitation. (B) STAT3 binds the PTTG promoter. Normalized inputs of HCT116 chromatin DNA were pulled down by STAT3 or negative IgG antibodies. DNA template was amplified by real-time PCR using primers 1-5. Human c-Fos promoter primers were used as positive controls and α-satellite repeat primers as negative controls. White bars: incubation with negative control IgG; black bars: incubation with STAT3 antibody. Each negative IgG control was normalized to unit “1”. Real-time PCR reaction was performed in triplicate and ChIP experiments repeated twice. (C) STAT3 activates PTTG transcription. HCT116 cells were co-transfected pGL 3 -Basic, PTTG promoter −2642/−1 or –1717/−1, together with empty vector pIRES2-ZsGreen1 (marked as ZsGreen, white bar), wild type STAT3 (grey bar) or STAT3-C (black bar), as described in Material and Methods, and luciferase activity measured. (D) STAT3 inhibitor S3I-201 suppresses PTTG transcription. HCT116 cells were transfected with pGL 3 -Basic, PTTG promoter −2642/−1 or –1717/−1, and treated by increasing amounts of S3I-201. Five ng of pRL-TK plasmid were co-transfected to normalize transfection efficiency. Transfections were performed in triplicate, and experiments were repeated three times. (E) STAT3 induces PTTG mRNA and protein expression. HCT116 cells were transfected with pIRES2-ZsGreen1 (marked as ZsGreen), STAT3, STAT3-C or STAT3-DN, and selected for stable transfectants. PTTG mRNA was assessed by real-time PCR (left), and STAT3 and PTTG protein were measured by Western blotting (right). Real-time PCR results are presented as mean ± SE. Western blots were quantified by Image-J and normalized to β-actin. Experiments were repeated three times.

Article Snippet: PTTG promoters, −2642/−1 and −1717/−1, were cloned into pGL 3 -Basic luciferase reporter vector (Promega, Madison, WI) as described ( ).

Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Incubation, Negative Control, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot