Synergy 2 luminometer

HIF-1α binding hypoxia-response element (HRE) and VEGF promoter containing luciferase vector (HRE-Luc and VEGF-Luc) were kind gifts from Dr. Jong-Wan Park (Seoul National University) [38 (link)]. For luciferase assay, luciferase and β-gal vector were transiently transfected into HEK293 or Caki-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and PolyFect transfection reagent (Qiagen, Venlo, Netherlands). After hypoxia or drug treatment in transfected cells, luciferase activities were measured using a Synergy 2 Luminometer (BioTek, Winooski, VT, USA) and normalized by β-gal assay.

To screen single compounds derived from natural products which attenuate β-catenin/TCF4 transcriptional activity, TOP and FOP flash luciferase plasmids (100 ng/mL) were transiently transfected into HEK293T cells with ectopic expression of pBABE-puro-LEF1 (1 μg/mL), pcDNA-Myc-TCF4 (0.5 μg/mL), and pcDNA3-β-catenin (0.5 μg/mL). The transfected HEK293T cells were seeded in 96-well culture dishes and incubated for 24 h to allow stabilization and protein expression. After incubation, cells were incubated with a natural compound library containing 502 single compounds at 5 μM of final concentration for 24 h. To measure β-catenin/TCF4 transcriptional activity in A375 melanoma cells, TOP and FOP flash luciferase plasmids (0.5 μg/mL) were transiently transfected using Lipofectamine 2000 without co-transfection of pBABE-puro-LEF1, pcDNA-Myc-TCF4, and pcDNA3-β-catenin. Luciferase activities were analyzed by using a Luciferase assay system (Promega, Madison, WI, USA) and Synergy 2 Luminometer (BioTek, Winooski, VT, USA), and then luciferase activities were normalized to the activity of β-galactosidase.

The Caspase-Glo 3/7 Assay (Promega) was performed according to manufacturer’s instructions. Briefly, purified B cells were plated in IMDM medium at the concentration of 100,000 cells/100 μl with stimuli [AffiniPure F(ab′)2 Fragment Goat Anti-Mouse IgM, μ Chain Specific (Jackson ImmunoResearch), mouse anti-CD40 (1 µg/ml, Becton Dickinson), and CXCL12 (25 nM, Peprotech)] for 24 h or left untreated. After stimulation, cells were counted and plated at 20,000/well in a white-walled multiwell plate. An equal volume of Caspase-Glo 3/7 Reagent was added to each well for 30 min. Luminescense signal was acquired on a Biotek Synergy 2 luminometer. Background readings were determined from wells containing culture medium without cells.

HCT-DK cells, that do not express miR-181a-5p, were seeded at a density of 80,000 cells/well in 24-well plates with complete McCoy's 5A supplemented with 10% fetal calf serum without antibiotics. The following day, cells were co-transfected with miR-181a-5p (both pMIR-REPORT plasmids -1000 ng/well- wild type or mutated for the miR-181a-5p seed site) or SCR precursor, and 100 ng/well of renilla luciferase control plasmid (pRL-TK; Promega, Madison, WI) using Lipofectamine LTX (Life Technologies,Madrid, Spain) according to the manufacturer's instructions. Luciferase assays were performed as previously described [40] .The enzymatic activities of renilla and firefly luciferases were quantified in a Synergy 2 luminometer (Biotek, Winooski, VT). Each combination of pMIR-REPORT (wild-type and mutated 3′UTR) and pRL-TK was tested in triplicate in five independent experiments. Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. The normalized data were expressed as changes relative to the data of the cells transfected with 100 nM miR-181a-5p mimic, SCR was taken as 100%.

Luciferase assay was performed as previously described [1 (link)]. HEK293T cells transiently transfected with steroid responsive element (SRE)-wild type (WT) or -mutant (Mut) containing luciferase vector (pSynSRE-T-Luc and pSynSRE-Mut-T-Luc) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). pSynSRE-T-Luciferase vectors (Addgene plasmid #60444 and #60490) were gifts from Timothy Osborne [66 ]. To screen the SREBP2 activating natural compounds, transfected HEK293T cells were incubated with a library of 502 natural compounds (20 μM of each compounds) for 24 h. Luciferase activities were analyzed by using a Synergy 2 Luminometer (BioTek, Winooski, VT, USA) and β-gal assay was used for normalization.