Dreamfect

MLE and A549 cells were transfected to produce high levels of telomerase and telomerase activity as we previously detailed [29 (link)]. 0.5×10⁶ cells were incubated with a mixture of 4 mg/ml liposome (Dreamfect, OZ Biosciences, Marseille, France) and 1 μg/ml plasmids containing TERT cDNA for 1 hr (kindly provided by Prof. Varda Rotter, Weizmann Research Institute), washed, cultured with adequate medium, incubated and analyzed at 24 hr.

Silencer Select siRNAs (siRNA-A, siRNA-B, negative control siRNA-1 and −2, Ambion, Life Technologies) were dissolved in 1 x siRNA buffer (Dharmacon, Lafayette, CO, USA). SiRNA-A and -B sequences are shown in Figure 1A. Transfections were performed according to the supplier’s protocol with 9 μl Dreamfect (OZ Biosciences, Marseille, France) at a final concentration of 50 nM siRNA within 1 ml culture medium that contained 5 × 10⁴ cells in a 12-well format (Greiner, Kremsmuenster, Germany). Transfection efficiency was measured by flow cytometry with fluorescently labeled control siRNA.
Experimental incubations lasted eight days with repeated transfections on day 0, 3, and 6. Prior to each transfection event, cell densities were determined by Cellscreen System (Innovatis, Bielefeld, Germany) on an Olympus IX 50 microscope (Olympus, Tokyo, Japan) and cells were subsequently reseeded at 5 × 10⁴ cells per ml.

ChIP assays were routinely performed using a pCMV-3XFLAG-ΔLf and pcDNA-hLf or a null vector and HEK293 cells which were transfected (1 µg of DNA for 1×10⁶ cells) using DreamFect (OZ Biosciences) [13] (link), [15] (link), [41] (link), [42] . The cells were lysed and sonicated using a BIORUPTOR to generate the chromatin preparation, and ChIP assays were performed using The MAGnify Chromatin Immunoprecipitation System kit (Invitrogen) according to the manufacturer's instructions. Chromatin was sonicated to an average size of 400 bp. A small fraction of the sonicated chromatin was put aside before the immunoprecipitation with antibodies, and constituted input DNA. ChIP complexes (2 10⁶ cells) were immunoprecipitated with anti-hLf (Sigma, St Louis, MT), anti-M2 (raised against the 3XFLAG present on 3XFLAG-ΔLf construction, Sigma), or anti-Rabbit IgG (GE Healthcare Life Sciences). The genomic DNA was purified and the recruitment of hLf and ΔLf proteins was measured by real-time qPCR, using specific primer pairs listed in Table S1. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. Amplification of the albumin promoter region was used as a negative control (data not shown) [15] (link).

For the luciferase assay experiments, two wells with 2×10⁵ HeLa cells per sample were seeded into 6-well plates. The next day 100 ng Renilla luciferase reporter, 20 ng Firefly luciferase reporter and 400 ng MS2 fusion protein per well were transiently transfected using DreamFect (OZ Biosciences) transfection reagent. One day later the cells from each sample were pooled and redistributed equally into 3 wells on a 6-well plate. The cells were harvested 48 h after transfection according to the Dual-Luciferase Reporter Assay System (Promega) technical manual. The luciferase measurements were subsequently performed according to the instructions in the technical manual on a Tecan Infinite M1000PRO microplate reader.

All chemical reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA) without additional purification. The OVA-modified and EGFP (5 moU) mRNAs, as well as DreamFect ™ (DF40500), were provided by OZ-Biosciences (Marseille, France). Before use, β-CD and choline chloride were dried to a constant weight in an oven at 75 °C.

Reporter gene assays were routinely performed in our laboratory using pcDNA-ΔLf or pcDNA-hLf constructs or a null vector and HEK 293 cells [15] (link), [41] (link), [42] . The reporter pGL3-SelH-Luc vector was obtained as in [13] (link) except that the 167 bp SelH promoter fragment was amplified with the primer pair listed in Table S1, cloned into the pGL3-promoter-Luc vector (Promega) and sequenced before use. HEK 293 cells were transfected (250 ng of DNA for 2×10⁵ cells, 50 ng of reporter vector and 200 ng of ΔLf, hLf expression vector or null vector) using DreamFect (OZ Biosciences, Marseille, France). Cell lysates were assayed using a luciferase assay kit (Promega) in a Tristar multimode microplate reader LB 941 (Berthold Technologies, Bad Wildbab, Germany). Relative luciferase activities were normalized to basal luciferase expression as described [13] (link) and expressed as fold increase to the relative luciferase activity of ΔLf or hLf. Basal luciferase expression was assayed using a null vector and was determined for each vector. Each experiment represents at least three sets of independent triplicates.