luciferase reporter gene plasmids pgl3-basic  (Shanghai GenePharma)

Journal: NPJ Breast Cancer

Article Title: Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1

doi: 10.1038/s41523-021-00300-1

Figure Lengend Snippet: a Endogenous AP1 activity in breast cancer cell lines analyzed by AP1 site-containing luciferase reporter plasmid. Data are means ± SD from three experiments. b BT549, MDA-MB-231, and MDA-MB-436 cells were treated with 5 µM U0126, SB203580, SP600125, or vehicle for 24 h followed by analyzing AP1 activity. Data are means ± SD from three experiments. * P < 0.001 vs vehicle; # P < 0.01 vs vehicle. c BT549, MDA-MB-231, and MDA-MB-436 cells were treated with 5 µM U0126, SB203580, or vehicle for 24 h followed by western analysis to detect JUN and GAPDH with the respective antibodies. Data are the representative of three independent experiments. All blots derived from the same experiment and were processed in parallel.

Article Snippet: AP1 transcriptional activity was measured with the aid of a firefly luciferase reporter gene plasmid (3xAP1pGL3) from Addgene (Watertown, MA).

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Western Blot, Derivative Assay

Journal: NPJ Breast Cancer

Article Title: Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1

doi: 10.1038/s41523-021-00300-1

Figure Lengend Snippet: a Luciferase-expressing MDA-MB-231 cells were transduced with lentiviral vector encoding either scramble or JAB1 shRNA for 3 days and then subcutaneously injected into nude mice. Tumor outgrowth was monitored weekly using the Xenogen IVIS-200 In Vivo bioluminescence imaging system. Data are means ± SD. n = 5. Statistical analysis was performed using Student t test (two-tailed) for the end experiment point. * P < 0.001. b Images of the xenograft tumors at 5 week. The image data is displayed in radiance or photons/sec/cm 2 /steradian. c Luciferase-expressing MDA-MB-231 cells were injected into nude mice for 5 days. Mice were randomized and treated daily with either Vehicle control or 1 µmol/kg of SB203580 i.p. for 4 weeks. Tumor growth were monitored weekly though Xenogen IVIS-200 in vivo imaging system. Data are means ± SD. n = 5. Statistical analysis was performed using Student’s t test (two-tailed) for the end experiment point. # P < 0.05. d Images of the xenograft tumors before treatment and after 4 weeks of treatment.

Article Snippet: AP1 transcriptional activity was measured with the aid of a firefly luciferase reporter gene plasmid (3xAP1pGL3) from Addgene (Watertown, MA).

Techniques: Luciferase, Expressing, Transduction, Plasmid Preparation, shRNA, Injection, In Vivo, Imaging, Two Tailed Test, Control, In Vivo Imaging

Journal: Experimental cell research

Article Title: The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene

doi: 10.1016/j.yexcr.2018.11.010

Figure Lengend Snippet: NME1 activates transcription of the ITGβ3 gene via direct binding to the promoter region. (a) Expression of mRNA encoding ITGβ3 was measured by quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) in the indicated melanoma (M14, WM1158, and WM793; −/+ forced NME1 expression) and mouse embryo fibroblast (wild-type C57BL/6, “WT”; NME1 knockout, “NME1ko”) cell lines. Expression of ITGβ3 in the respective control conditions of each panel (vector or WT) is normalized to a value of 1. (b) Shown at top is a schematic representation of a promoter-reporter cassette containing the ITGB3 promoter (“ITGB3-P”; −301 to +13) in linkage with a firefly luciferase reporter mini-gene. Summarized below are amounts of luciferase activity obtained after transient transfection of the indicated melanoma cell lines with the ITGB3 promoter-luciferase plasmid in the absence or presence of forced NME1 expression. Activity is expressed as relative luciferase activity (“Rel. luc. activity”), with activity obtained in the absence of forced NME1 expression assigned a value of 1. *Denotes p < 0.05 by Student’s t-test. (c). Impact of forced NME1 expression on occupancy of the ITGB3 promoter by transcriptionally active chromatin and NME1 was assessed in M14 cells by chromatin immunoprecipitation assay (ChIP). ChIP reactions were conducted with antibodies directed to IgG, acetylated histone 3 (at lysine 27, or “H3K27ac”), or NME1 as indicated. Immunoprecipitated DNA was analyzed by qPCR with a series of five amplicons (150– 200 bp) spanning the ITGB3 promoter from −−958 to +15, as shown. *denotes p < 0.05 by Student’s t-test.

Article Snippet: The ITGβ3 promoter-luciferase reporter plasmid was generated by PCR amplification of the −301 to +13 region from human genomic DNA, followed by purification and ligation of the fragment into the pGL3-Basic (promoter-less) plasmid containing a firefly luciferase reporter mini-gene (Promega, Madison, WI) with T4 ligase (New England Bioscience, Ipswich, MA).

Techniques: Binding Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knock-Out, Control, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation, ChIP-chip, Immunoprecipitation