Renilla luciferase expression plasmid

293T HEK cells in 24-well plates were transfected with a total amount of 1 µg DNA by Attractene. A renilla luciferase expression plasmid (Promega, Madison, WI, USA) as internal control, a luciferase reporter plasmid driven by a 1.7 kb Puma promotor region (−1200 to +500 from ATG) containing a conserved binding site for FOXO3A (CAAACAAT or mutated to CAGGGAAT)^{25 (link)} and FOXO3A-TM (T32A, S253A, S315A) pcDNA3.1 expression plasmid were used in a 1:1:1 ratio. Empty vector was added as needed to keep the total DNA constant. Eight hours after transfection DMSO or CT98014 (0.75 µM) were added. Cells were lysed using 100 µl luciferase-lysis buffer (50 mM Tris-phosphate pH 7.8, 250 mM KCl, 10% Glycerol, 0.1% NP-40), lysates cleared by centrifugation at 16.100×g for 5 min, 4 °C. A volume of 10 µl of the lysate was added in two black 96-well plates, renilla buffer (25 mM Tris-phosphate, 100 mM NaCl, 1 mM EDTA, 0.05 mM Coelenterazin) or luciferase buffer (25 mM Tris-phosphate, 10 mM MgSO₄, 2 mM ATP, 0.05 mM Luciferin) were auto-injected and firefly and renilla luciferase activities were determined by a plate reader.

HT-29 and HGUE-C-1 cells were seeded in 24-well culture plates (180,000 and 150,000 cells/well, respectively). When cells reached 40–60% confluence (24 hr), they were transiently transfected with 0.8 μg of pRb-TA-Luc or pE2F-TA-Luc responsive luciferase reporter plasmid (Clontech, Mountain View, CA), using Lipofectamine2000 reagent (Invitrogen, Carlsbad, CA, USA) in a DNA transfection agent ratio of 1:3. The pTa-Luc vector lacking the response element was used as negative control. Six hours after transfection, culture medium was replaced by fresh medium and cells were kept under normal growth condition in DMEM with 10% foetal serum for 24 hours prior to lysis and luciferase detection. All transfections were performed as co-transfections using a Renilla luciferase expression plasmid (Promega Corporation, Madison, WI) to establish an internal control for transfection efficiency. The transfected cells were washed once with PBS and lysed using 100 μl Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions, using for detection of the chemiluminescent signal, a luminometer from Berthold (Montreal Biotech, Kirkland, QC). Promoter activities of the RB and E2F reporter plasmids were expressed using the arbitrary units “RLU” (relative luciferase units).

Luciferase reporter plasmids were constructed by cloning a 2-kb-long fragment centered around the TSS upstream of the firefly luciferase gene in a pGL3-basic vector (Promega) for each promoter or a variant fragment generated by PCR mutagenesis of the putative CDE/CHR motif. NIH/3T3 cells (10⁶ cells) were transfected using Lipofectamine 2000 with 3 µg of the luciferase reporter plasmid and 30 ng of Renilla luciferase expression plasmid (pGL4.73; Promega) for normalization and treated or not with 10 µM nutlin 3a. Transfected cells were incubated for 24 h, trypsinized, resuspended in 75 µL of culture medium with 7.5% fetal calf serum, and transferred into a well of an optical 96-well plate (Nunc). The Dual-glo luciferase assay system (Promega) was used according to the manufacturer's protocol to lyse the cells and read firefly and Renilla luciferase signals. Results were normalized, and the average luciferase activity in cells transfected with a wild-type promoter and not treated with nutlin was assigned a value of 1. Differences between two groups were analyzed by Student's t-test, and values of P ≤ 0.05 were considered significant.