Renilla glo luciferase assay

MB-231 reporter cells were transfected with a negative control (NC) RNA, PM-miR-34a, or FM-miR-34a at the indicated concentrations using Lipofectamine RNAiMAX (Life Technologies). At each time point, Renilla-Glo Luciferase assay (Promega) was performed as per manufacture instructions. In brief, Renilla-Glo Luciferase substrate was mixed with Renilla-Glo buffer at 1:1000 dilution followed by addition into each well. After shaking the plates at room temperature for 10 min, Renilla luciferase signal was measured using a GloMax plate reader (Promega). In the case of Renilla luciferase assay following Ago2 knockdown, MB-231-miR-34a sensor cells were seeded in individual wells of a 96-well plate. The following day, cells were co-transfected with 50 nM siRNA against Ago2 (GeneSolution GS27161; QIAGEN) or a control siRNA (4390846; Thermo Fisher Scientific) along with 10 nM NC, PM-miR-34a, FM-miR-34a duplexes, or miR-34a mimic (MC11030; Ambion) using Lipofectamine RNAiMAX (Life Technologies). Renilla luciferase assay was performed as described above 48 h post-transfection.

MDA-MB-231-miR-34a reporter cells or LNCaP-miR-34a sensor cells were treated with 100 nM miR-34a duplex, 100 nM folate-NC, and 100 nM folate–miR-34a (hereafter referred to as folate-34a). At 72 h, Renilla-Glo Luciferase assay (Promega, Madison, WI, USA) was performed as per manufacturer instructions. In brief, Renilla-Glo Luciferase substrate was mixed with Renilla-Glo buffer at 1:1000 dilution followed by addition into each well. After shaking the plates at room temperature for 10 min, Renilla luciferase signal was measured using a BioTek Synergy microplate reader (Agilent, Santa Clara, CA, USA).

HEK-293T were plated in 24-well plates (5 × 10⁵ cells per well). One day later, HEK-293T cells were co-transfected (JetPRIME; Polyplus) with IFN-β-pGL3 or pISRE-Luc plasmid (0.3 μg/well; Stratagene) that contain the firefly luciferase reporter gene downstream of an IFN-β-specific promoter sequence or the ISRE enhancer element upstream, respectively. Cells were simultaneously co-transfected with the pRL-CMV reference plasmid (0.03 μg/well; Promega) and the empty pCI-neo-3×FLAG expression vector (0.3 μg/well) or encoding proteins as specified. When specified, cells were transfected with 0.1 μg/well of poly(dA:dT) (Invivogen) or treated with 1 x 10³ IU/ml of recombinant IFN-β (PBL Assay Science) 24h after transfection. After 24h post-IFN-β-stimulation or 48h post-transfection, the cells were lysed (Passive lysis buffer, Promega), and both firefly and Renilla luciferase activities in the lysate were determined using the Bright-Glo and Renilla-Glo luciferase assay systems (Promega), respectively. The reporter activity was calculated as the ratio of firefly luciferase activity to the reference Renilla luciferase activity. All graphs show mean values and include error bars indicating the standard deviations (SD).