ap-1 luc  (Beyotime)

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Effects of specific inhibitors on p38 and AKT in CD46-mediated MMP9 promotion. (A) RT-qPCR of total RNA from 5637 cells to assess CD46 and MMP9 expression levels. (B) Transfection of bladder cancer cells with either AP-1-luc or MMP9-luc plasmids, followed by reporter gene transcription analysis. (C) UM-UC-3 cells underwent a reporter gene transcription assay to evaluate the effect of p38 (SB202190) and AKT (LY294002) inhibitors on CD46-mediated transcriptional activities of AP-1 (left panel) and MMP9 (right panel). (D) Treatment of both 5637 and J82 cells with indicated inhibitors for 24 h, followed by western blot analysis. Densitometric analysis of MMP9 expression is displayed as bar graphs at the bottom of each blot. Each bar represents mean ± SD. Statistical differences between groups were determined using a two-tailed unpaired Student's t-test for A and B and two-way ANOVA with Bonferroni multiple comparisons test for C and D. * P<0.01, ** P<0.001, *** P<0.0001 vs. control cells. veh, vehicle; AKT, protein kinase B; MMP9, matrix metalloproteinase 9; RT-qPCR, reverse transcription-quantitative PCR; AP-1, activator protein 1.

Article Snippet: The AP-1-luc vector was obtained from Stratagene (Agilent Sumitomo Dainippon Pharma Co., Ltd.).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Transcription Assay, Western Blot, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9

doi: 10.3892/ijo.2024.5659

Figure Lengend Snippet: Model of CD46-induced MMP9 upregulation in bladder cancer cells. This figure proposes a model for CD46-mediated promotion of bladder cancer metastasis. Transmembrane receptor CD46 activates hyperphosphorylation of p38 MAPK and PI3K/AKT, leading to c-Jun activation in the AP-1 complex at the MMP9 promoter. Increased MMP9 secretion into the extracellular matrix enhances migration and invasion of bladder cancer cells. Secreted MMP9 may also cleave the extracellular domain of CD46, potentially regulating overstimulation of CD46-mediated MMP9 expression. The model was created using BioRender.com . MMP9, matrix metalloproteinase 9; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; AP-1, activator protein 1.

Article Snippet: The AP-1-luc vector was obtained from Stratagene (Agilent Sumitomo Dainippon Pharma Co., Ltd.).

Techniques: Activation Assay, Migration, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: IgG immune complex-induced acute lung injury is ameliorated by cAMP via down-regulation of C/EBP- and AP-1-mediated transcriptions

doi: 10.1186/s12950-023-00359-6

Figure Lengend Snippet: AP-1- and C/EBP-mediated transcriptions are reduced by cAMP. HEK293/RAW264.7 cells are transfected with the indicated plasmids. 24 h later, HEK293 cells are treated with DMSO or Rolipram (10 µM) for 6 h. RAW264.7 cells are pre-treated with DMSO or Rolipram for 1 h. Then the cells are treated with or without 100 µg/ml of IgG-IC for 6 h. The cells are lysed, and subjected to luciferase assays. Data are expressed as mean ± SEM (N = 3). *, ** and *** suggest p < 0.05, 0.01 and 0.01, respectively

Article Snippet: HEK293/RAW264.7 cells are transfected with AP-1 Luc (Beyotime Biotechnology) plus TK (Promega) or C/EBP Luc (Promega) plus TK by using Fugene®6 from Promega.

Techniques: Transfection, Luciferase

Journal: Cancers

Article Title: EF-24, a Curcumin Analog, Inhibits Cancer Cell Invasion in Human Nasopharyngeal Carcinoma through Transcriptional Suppression of Matrix Metalloproteinase-9 Gene Expression

doi: 10.3390/cancers15051552

Figure Lengend Snippet: EF-24 suppresses MMP-9 gene transcription by interfering with nuclear translocation of NF-κB. HONE-1 cells expressing a luciferase gene driven by wild-type ( A ) and various mutated forms ( B – E ) of MMP-9 promoter were pretreated with EF-24 for 1 h and then incubated with TPA for an additional 23 h. Luciferase assays were used to measure the transcriptional activity of MMP-9 promoter. Schematic representations of the reporter plasmids containing a mutated SP-1 ( B ), AP-1 ( C , D ), or NF-κB responsive element ( E ) are shown on the top. To explore the nuclear translocation of NF-κB, a nuclear extract of untransfected HONE-1 cells after the treatment of EF-24 and TPA was isolated and analyzed for the protein levels of NF-κB ( F ). Blots are representative of three independent experiments. Densitometric analyses were conducted by ImageJ, and quantitative results were normalized to the nuclear levels of an internal control, lamin B2. In addition, HONE-1 cells treated with EF-24 and TPA were fixed and stained with DAPI and a specific antibody against NF-κB p65 ( G ). The interaction of NF-κB with MMP-9 promoter was analyzed by using chromatin immunoprecipitation assay (ChIP) with an NF-κB antibody in HONE-1 cells treated with or without NF-24 and TPA ( H ). Densitometric analyses were conducted by ImageJ. a Significantly different, p < 0.05, when compared with the control group. b Significantly different, p < 0.05, when compared with the TPA treatment group. c Significantly different, p < 0.05, when compared with the TPA treatment plus EF-24 (0.25 μM) group. d Significantly different, p < 0.05, when compared with the TPA treatment plus EF-24 (0.5 μM) group.

Article Snippet: The constructs containing NF-κB-Luc, SP-1-Luc, or AP-1-Luc sequences were obtained from Stratagene (La Jolla, CA, USA).

Techniques: Translocation Assay, Expressing, Luciferase, Incubation, Activity Assay, Isolation, Staining, Chromatin Immunoprecipitation