Rela reporter construct

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.