Cignal finder reporter array

A549 cells were reverse-transfected with Mimic/Inhibitor control and miR-494-3p Mimic/Inhibitor (26 nM) on a 96-wells plate pre-coated with reporter genes for 10 transcription factors (TF), as well as positive and negative controls (Cignal Finder Reporter array, Signal Transduction 10 Pathways, SaBiosciences Corp., Frederick, MD; Supplementary Table 9) as described [17 (link)]. Cells were harvested after 72 h and dual luciferase emission was assessed using a Dual-Glo Luciferase Assay (Promega Corporation, Madison, WI, USA) and a luminometer. Firefly luciferase emission was normalized on Renilla for each sample. Values of negative controls were used to set the threshold for TF activity. Relative pathway activation was calculated in miR-494-3p over expressing cells compared to control and a p-value < 0.05 were set as cut-off for significant difference.

Cignal Finder Reporter Array (336821, Qiagen/SABiosciences, Frederick, MD, USA) was used to assess 45 different signaling pathways. HeLa cells were seeded into wells (50,000 cells/well) of the Cignal Finder 96-well plates (CCA-901L, Qiagen, SABiosciences, Frederick, MD, USA) for introducing pathway reporters into cells by reverse transfection according to the manufacturer’s protocol. Briefly, reporter DNA constructs in each plate well were re-suspended with 50 μL Opti-MEM and then mixed with 50 μL diluted Lipofectamine 2000 transfection (Life Technologies, Carlsbad, CA, USA) reagent. Cells were suspended in Opti-MEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% of FBS and 0.1 mM non-essential amino acids at a density of 6 × 10⁵ cells/mL, and then 50 μL of the cell suspension was added into each plate well and mixed with DNA resident in the plate and added transfection reagent. The cells were incubated for 3 h. Following transfection; the cells were treated with vehicle (Opti-MEM) or 100 nM FD-895 for 3 h in Opti-MEM media. Luciferase and renilla expression were determined (Qiagen/SABiosciences Corp., Frederick, MD, USA).