Journal: Journal of Translational Medicine

Article Title: NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug

doi: 10.1186/1479-5876-10-252

Figure Lengend Snippet: TMZ increases NF-κB transcriptional activity in MMR-proficient but not in MMR-deficient cells. ( A ) The cells were transiently co-transfected with the pNF-κB-Luc and pRL-null vectors, and 24 h later exposed to 50 μM TMZ+10 μM BG or to BG alone. Firefly luciferase activity was determined 48 and 72 h after drug treatment and normalized to that of Renilla luciferase and then to the total amount of protein used in the assays. Data are expressed in terms of fold change (i.e. the ratio between firefly luciferase activity/μg protein detected in TMZ-treated cells and that detected in the corresponding control cells). Values represent the mean of at least four independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). * p<0.05, according to paired Student’s t test analysis performed comparing firefly luciferase activity/μg protein of TMZ-treated samples with that of the corresponding controls. ( B ) M10 cells were cultured in the presence of 50 μM TMZ+10 μM BG or BG alone and culture supernatants were collected after 48 and 72 h of incubation. IL-8 and MCP-1 amount in the culture supernatants was then determined by ELISA and expressed in terms of pg protein/10 6 cells. Values represent the mean of three independent experiments performed with duplicate samples. Bars, SEM. ** p<0.01 and * p<0.05, according to Student’s t test analysis performed comparing the chemokine amount detected in the culture supernatants of TMZ-treated cells with that detected in the culture supernatants of the corresponding controls.

Article Snippet: HCT116/3-6, HCT116, M10, pUSE2 and KD12 cells were seeded into 24-well plates (BD Falcon™, Becton, Dickinson and Company, Franklin Lakes, NJ) and allowed to adhere at 37°C for 18 h. The cells were then transiently co-transfected with 3 μg (M10) or 0.075 μg (HCT116/3-6, HCT116) or 0.3 μg (pUSE2 and KD12) of the NFκB-responsive firefly luciferase reporter pNF-κB-Luc (Stratagene, La Jolla, CA) and 250 ng (M10) or 6.25 ng (HCT116/3-6, HCT116) or 25 ng (pUSE2 and KD12) of the Renilla luciferase expression vector pRL-null (Promega Corporation, Madison, WI) using Lipofectamine™ 2000 Reagent (Invitrogen Corporation) according to the manufacturer’s protocol.

Techniques: Activity Assay, Transfection, Luciferase, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: A single cell cycle genes homology region (CHR) controls cell cycle-dependent transcription of the cdc25C phosphatase gene and is able to cooperate with E2F or Sp1/3 sites

doi:

Figure Lengend Snippet: NF-Y is the main activator for cdc25C transcription. Increasing amounts of dominant-negative NF-YA subunit down-regulate the wild-type cdc25C luciferase reporter construct. Firefly luciferase reporter m25C-wt-luci, together with pRL-null plasmid expressing Renilla luciferase control, was transfected in NIH 3T3 cells. Increasing amounts of a plasmid coding for a dominant-negative form of NF-YA, YA13m29, were co-transfected. Firefly luciferase activity was normalized to Renilla luciferase. The value for transfections without YA13m29 was set at 100% and other activities are given relative to that. Three independent experiments were carried out with three assays per experiment and standard deviations are shown.

Article Snippet: Transfections were carried out in 4-fold using 6 µl Pfx-3 (Invitrogen, Leek, The Netherlands) with 2 µg luciferase reporter construct and 40 ng of pRL-null vector (Promega) in 1 ml Optimem transfection medium (Gibco, Eggenstein, Germany) per well.

Techniques: Dominant Negative Mutation, Luciferase, Construct, Plasmid Preparation, Expressing, Transfection, Activity Assay