Luminometer

VSMCs were seeded 48 h before transfection in 24 well plates at a concentration of 2x10⁴ cells per plate in DMEM (2% glutamine, 1% penicillin-streptomycin, 10% SVF). At 70% confluence cells were washed 20 min before transfection. The transfection mixture was as follow: 400ng of luciferase reporter DNA [-1153; +46]sPLA2-Luc or multimeric-NF-κB[(Ig-κB]-Luc already described, 100ng of CMVβ-galactosidase, 1.6μl of Lipofectamine plus and Lipofectamine (Invitrogen) in 200μl of OPTIMEM (Sigma) and for transactivation studies 10ng of pCMV-AMPKα2 catalytic and negative dominant expression vector were added to the mix. The cells were refed 3h later with 200μl of DMEM containing 5% SVF for 1h and then the culture medium was replaced by DMEM containing 0.2% SVF. Twenty hours later after the start of the transfection, the medium was changed and the cells were pretreated with indicated concentration of AICAR (5-aminoimidazole-4-carboxamide-1-β–D-riboside), phenformin (phenylbiguanide) for 4h, then stimulated with IL-1β (10ng/ml) and the incubation was continued for a further 24h. Luciferase activity was measured using luciferase reporter assay kit (PROMEGA), with signal detection for 12s by a luminometer (Berthold, Pforzheim, Germany) and normalized by dividing the relative light units by β-galactosidase activity. The degree of induction was calculated relative to the control (minus IL-1β).

The human collagen 1A1 promoter constructs spanning -2300/+18 and -205/+18, respectively, linked to a luciferase reporter gene in pGL3 basic (Promega, Mannheim, Germany) were a kind gift from Annett Skupin [16 (link)]. Constructs were transfected into subconfluently grown human skin fibroblasts by lipofection (Lipofectamin reagent 2000, Invitrogen). In order to standardize transfection efficacy cells were co-transfected with a humanized Renilla luciferase vector (phRL, Promega). Cells were treated with 10% milk or 10 ng/ml TGFβ1 for 48 hours. Finally, cells were lysed and luciferase activities of both luciferases were detected separately, using the Dual-Luciferase Reporter Assay System (Promega) and a luminometer (Berthold, Bad Wildbad, Germany).

The luciferase reporter constructs under the human p15 promoter (-2.5 kb ∼ +0.16 kb) and p21 promoter (-2.4 kb ∼ +0.01 kb) were kindly provided by Prof. Ye-Guang Chen (Tsinghua University, Beijing, China). HEK 293T cells were seeded at a density of 8×10⁴ cells per well in 24 well plates and transfected with various amounts of plasmids as indicated in the figures. Transient transfection was performed with VigoFect (Vigorous, Beijing, China). 48 h after transfection, the cells were harvested and luciferase activities were measured by a luminometer (Berthold Technologies, Stuttgart, Germany). The internal control Renilla activity was used to normalize the luciferase activity. Each assay was performed in triplicate and the data represent the mean ± s.e. of three independent experiments.

The MH7A cells were transfected with pGL3-hBAFF-Luc and pcDNA-lacZ for monitoring transfection efficiency by β-galactosidase assay. Luciferase activity was determined by incubating cell extracts with luciferase substrate (Promega). Luminescence was measured using luminometer (Berthold Technologies, Oak Ridge, TN, USA). Luciferase units of experimental vector were normalized to the control vector in each sample.^{31 (link), 32 (link), 48 (link)}

Cells were transfected and HDACi-treated as described above. To assess for transfection efficiency, cells were transfected in parallel with peGFP-NLS. A transfection efficiency of 40% to 60% (assessed by visual inspection of the cells under the fluorescent microscope) was routinely achieved. Luciferase activity was measured 24 hours post-transfection using the Luciferase Assay System (Promega). Briefly, 100 μL Luciferase Assay Reagent was injected into 20 μL cell lysate per transfected sample. Relative Luminescence Units (RLU) were measured for 60s using a luminometer (Berthold Technologies, Germany). Luciferase activity was normalized to the total protein content of the cell lysates, as determined by the Bradford reagent (Sigma-Aldrich). The results shown are representative of at least three different experiments using different DNA preparations.

TCF/LEF activity was determined using a Cignal TCF/LEF Reporter Assay kit (luc) (Qiagen). CLDE cells were transfected with a TCF/LEF reporter plasmid, then cultured with Ca²⁺ for 24 h and stimulated with Wnt3a for 24 h, after which luciferase activity was determined using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) with a luminometer (Berthold Technologies, Oak Ridge, TN, USA). The activity was normalized to that of Renilla luciferase, which served as the internal control.