Centro xs lb960 microplate luminometer

The transcriptional regulation of MMP-9 was examined by the transient transfection of an MMP-9 promoter–luciferase reporter construct (pGL4-MMP-9). The plasmid pGL4-MMP-9 promoter (spanning nucleotides from −925 to +13) was kindly provided by Dr. Young-Han Lee (Konkuk University, Korea). The NF-κB and AP-1 luciferase reporter plasmid were purchased from Clontech (Palo Alto, CA, USA). The effects of sulforaphane on MMP-9 promoter activity were determined by pretreating cells with sulforaphane for 1 h prior to the nicotine treatment. Cells were collected with cell culture lysis reagent (Promega, Madison, WI, USA) and the luciferase activity was determined using a luminometer (Centro XS lb960 microplate luminometer, Berthold Technologies, Oak Ridge, TN, USA) according to the manufacturer’s protocol.

Plasmid pGL4-MMP-9 promoter (spanning nucleotides from -925 to +13) was kindly provided by Dr. Young-Han Lee (Konkuk University, Korea). SW620 cells were seeded and grown until they reached 70%-80% confluence. The pGL4-MMP-9 promoter plasmid was then transfected into cells using FuGENE 6 (Promega, Madison, WI, USA) according to the manufacturer’s protocol. PRL-TK, an internal control plasmid containing constitutively active Renilla luciferase reporter gene linked to the promoter of herpes simplex thymidine kinase, was transfected as an internal control. Cells were incubated with the transfection medium for 1 day and then treated with CA for 6 h. Co-transfection studies were performed in the presence or absence of the expression vector encoding the dominant-negative mutants MEK-1 (pMCL-K97M), JNK (pMCL-TAM67), or p38 MAPK (pMCL-mP38), kindly provided by Dr. N.G. Ahn (University of Colorado-Boulder, CO), Dr. M.J. Birrer (NCI, Rockville, MD, USA), and Dr. J. Han (Scripps Research Institute, CA, USA), respectively. These cells were harvested with a cell culture lysis reagent (Promega, Madison, WI, USA). Luciferase activities were determined using a luminometer (Centro XS LB960 microplate luminometer, Berthold Technologies, Germany) according to the manufacturer’s protocol.

Analysis of NF-kB activity was performed by cloning six canonical NF-kB RELA (p65) binding sites upstream of the luciferase gene in the pGL3-basic reporter vector. MDA-MB-231 cells overexpressing CTCF were transfected with the luciferase reporter and pRL-TK plasmid. After 48 h, cells were lysed with passive lysis buffer and the lysates were analyzed for both firefly and Renilla luciferase activity using a dual-luciferase reporter assay kit (Promega). Luciferase activity was normalized for transfection efficiency using the pRL-TK reporter (Promega) as an internal control. A Centro XS³ LB 960 Microplate Luminometer (Berthold) was used according to the manufacturer’s instructions. The results are expressed as the percentages of relative luciferase activities of the control, which was set as 100%.

PlncRNA-1 wild type (WT), PlncRNA-1 mutation type 1 (MT 1), and PlncRNA-1 mutation type 2 (MT 2) were constructed by Genomeditech (Shanghai, China) and co-transfected into 293T cells along with the miR-136-5p mimic or miRNA controls using the Lipofectamine 3000 kit (Invitrogen). The cells were incubated for 48 h after transfection. Dual-luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega, WI, USA), following the manufacturer’s instructions. Luciferase functions were measured using Centro XS³ LB 960 Microplate Luminometer (Berthold Technologies, USA), following normalization by renilla luciferase.

Equal volumes of detached confluent cultures were analysed using Nano-Glo® Luciferase Assay [Promega #N1110] and the reader Centro XS³ LB 960 Microplate Luminometer [Berthold Technologies], using the software MikroWin 2010 version 5.15 (Mikrotek Laborsysteme GmbH, Germany).

The Gaussia luciferase assay was performed 72h after transduction by transferring 20μl of cell culture supernatant into a black luminometer plate (NUNC) [19 (link)]. The substrate was prepared by 1:1,000 dilution of 2mM coelenterazine (P.J.K.) in assay buffer (1.1M NaCl, 220mM K₂HPO₄/KH₂PO₄, 0.44mg/ml BSA, 1.3mM NaN₃, pH 5). Centro XS³ LB 960 Microplate Luminometer (Berthold, Bad Wildbad, Germany) was used to inject 100μl of the substrate, and measurement of relative light units was performed for 1sec after a delay of 1sec after injection for each individual well. Background measurements of cell culture media removed directly after transduction were subtracted from measurements 72h after transduction.
Firefly luciferase assay was performed 72h after transduction by lysing the cells for 2mins by adding Bright-Glo reagent (Promega). The measurement was carried out using a Centro XS³ LB 960 Microplate Luminometer within 5min after addition of the reagent.
When GFP was used as reporter, GFP-positive cells were counted 72h after transduction and transducing units per ml were calculated.