Renilla luciferase flash assay kit

A luciferase assay was used to determine possible interactions between KLF5 and NFkB, as previously described (Lim et al. 2013a) . Primary myometrial cells, prepared as described previously, at w70% confluence, were transfected with 0.75 ng NFkB reporter construct (Qiagen) using FuGENE HD Transfection Reagent (Promega). After 6 h, cells were transfected with 200 nM KLF5 or NC siRNA (as detailed above) for 48 h. The medium was then replaced with DMEM/F-12 (containing 0.5% BSA), with or without 1 ng/ml IL1b, 5 mg/ml poly(I:C) or 1 mg/ml flagellin, and the cells were incubated at 37 8C for an additional 24 h. The cells were harvested in lysis buffer, and luminescence activity was measured using the Luciferase Reporter Assay Kit (Life Research, Scoresby, VIC, Australia) and Renilla Luciferase Flash Assay Kit (Thermo Fisher Scientific, Scoresby, VIC, Australia) as instructed. The ratio of the firefly luciferase level to the Renilla luciferase level was determined and the results are expressed as a ratio of normalised luciferase activity of IL1b-, poly(I:C)-or flagellinstimulated NFkB reporter plus NC siRNA transfected cells, which was set as 1. The experiments were performed from myometrium obtained from five patients.

A luciferase assay was used to determine possible interactions between GSK3b and NFkB, as described previously (Lappas 2013 (link), Lim et al. 2013a) , with some modifications. For these studies, primary myometrial cells, prepared as described previously, at w70% confluence were transfected with the FuGENE HD Transfection Reagent (Promega) according to the manufacturer's guidelines. The cells were co-transfected with 0.15 mg NFkB reporter construct (Qiagen) plus 200 nM NC siRNA or GSK3b siRNA for 48 h. The medium was then replaced with DMEM/F-12 with 0.5% BSA, with or without 1 ng/ml IL1b or 10 ng/ml TNFa, and the cells were incubated at 37 8C for an additional 24 h. The cells were harvested in lysis buffer, and the luminescence activity was measured using the Luciferase Reporter Assay Kit (Life Research) and Renilla Luciferase Flash Assay Kit (Thermo Fisher Scientific, Scoresby, VIC, Australia) as instructed. The ratio of the firefly luciferase level to the Renilla luciferase level was determined and the results are expressed as a ratio of normalised luciferase activity of IL1b-or TNFa-stimulated NFkB reporter plus NC siRNAtransfected cells, which was set as 1. The experiments were performed from the myometrium obtained from five patients.

A luciferase assay was also used to determine the effect of siPARK7 on NF-κB RELA transcriptional activity as previously described (Lim et al. 2016b ). Briefly, primary myometrial cells were transfected with 300 ng/mL RELA reporter construct (Qiagen) using FuGENE HD transfection reagent (Promega). After 6 h, cells were transfected with 50 nM of siPARK7 or siCONT (as detailed above) for 48 h. The medium was then replaced with DMEM/F-12 (containing 0.5% BSA), with or without 1 ng/mL IL1B, 10 ng/mL TNF, 250 ng/mL fsl-1, 1 μg/mL flagellin or 5 μg/mL poly(I:C), and the cells were incubated at 37°C for an additional 20 h. After final incubation, cells were harvested in lysis buffer, and luminescence activity was measured using a luciferase reporter assay kit (Life Research; Scoresby, Vic, Australia) and Renilla luciferase flash assay kit (Thermo Fisher Scientific) as instructed. The ratio of the firefly luciferase level to the Renilla luciferase level was determined and the results are expressed as a ratio of normalised luciferase activity. The experiments were performed from myometrium obtained from six patients.

To determine the effect of siIRF5 on RELA transcriptional activity, a luciferase assay was performed as previously described (Lim et al. 2016b) . Briefly, primary myometrial cells were transfected with 300 ng/mL RELA reporter construct (Qiagen) using FuGENE HD transfection reagent (Promega). After 6 h, cells were transfected with 50 nM of siIRF5 or siCONT (as detailed above) for 48 h. The medium was then replaced with DMEM/F-12 (containing 0.5% BSA), with or without 1 ng/mL IL1B, 10 ng/mL TNF, 250 ng/mL fsl-1 or 1 μg/ mL flagellin and the cells incubated at 37°C for an additional 20 h. After final incubation, cells were harvested in lysis buffer, and luminescence activity was measured using a luciferase reporter assay kit (Life Research; Scoresby, Vic, Australia) and Renilla luciferase flash assay kit (Thermo Fisher Scientific) as instructed. The ratio of the firefly luciferase level to the Renilla luciferase level was determined, and the results are expressed as a ratio of normalised luciferase activity. The experiments were performed from myometrium obtained from six patients.