Enspire 2300 luminometer

The luciferase assay was performed as previously described [12 (link)]. Briefly, MCF7 and MDA-MB-231 cells were transiently co-transfected with a firefly luciferase construct (100 ng) containing the Transcriptional Responsive Elements (TREs) of selected genes and the Renilla luciferase vector (10 ng) to normalize transfection efficiency (Cignal Finder Reporter assay kit, QIAGEN, Hilden, Germany). A non-inducible reporter construct encoding firefly luciferase under the control of a basal promoter element (TATA box), without any additional TREs, and a constitutively expressing GFP construct were used as negative and positive controls, respectively. After 24 h from transfection, cells were treated with SR and CBD (5 μM) for 18 h. Subsequently, the efficiency of transfection was evaluated in cells transfected with the positive control, and luciferase activity was measured using a dual luciferase assay system (Promega, Madison, WI, USA). Luciferase readings were measured using an EnSpire-2300 luminometer (Perkin Elmer, Waltham, MA, USA). Data were represented as relative luciferase activity, obtained by the ratio of firefly values (promoter reporter) to Renilla values (control reporter). Experiments in triplicate were repeated at least three times, and values were expressed as the mean ± SD.

Cells were transiently cotransfected with the TCF/Lef firefly luciferase construct (100 ng) and the Renilla luciferase vector (10 ng) (Cignal^TM TCF/LEF Reporter (GFP) assay; QIAGEN). Luciferase activity was measured using a Dual-luciferase assay system (Promega) and an EnSpire-2300 luminometer (Perkin Elmer). Relative firefly luciferase activity was obtained by normalizing it to that of Renilla luciferase activity.

The EBs were transferred from the conical bottom 96-well plate to a white 96-well plate for luminescence measurements. For this, 150 μl OptiMEM was added per well to dilute the differentiation medium and the EBs transferred in a volume of 50 µl to the white plate using a multichannel pipette. Luminescence was measured using the Promega Nano-Glo Live Cell Assay System (Promega, Madison, USA) according to the manufacturer’s instructions with optimized conditions: Nano-Glo Luciferase Assay Substrate was diluted 1:40 in Nano-Glo Luciferase Assay Buffer. Subsequently, one volume of Opti-MEM I Reduced Serum Medium, no phenol red (Thermo Fisher Scientific) was added, and 25 μl of this diluted substrate added per well of the white plate. The plate was centrifuged briefly and luminescence measurements performed 8 min after substrate addition on a PerkinElmer Enspire 2300 luminometer.