Cignal 45 pathway reporter array

The Cignal 45-Pathway Reporter Array was purchased from Qiagen (Cat#: CCA-901L). The array assays were essentially carried out according to the protocol recommended by the manufacture. Reverse transfection was performed using HepG2 cells (8x10⁴ cells/well in 98-well plates). Sixteen hours post-transfection, transfected cells were treated with CDCA (25 μM), CA (25 μM) or vehicle DMSO for 30 hours, followed by detection of luciferase activities by the Dual Luciferase Assay [36 (link)].

The firefly luciferase reporter constructs superTOPflash and superFOPflash (kindly provided by Konrad Basler, UZH Zürich) were used to quantify Wnt/β-catenin-mediated transcriptional output. Cells were plated in duplicates in 24-well plates and transfected the following day with 500 ng of the superTOPflash or superFOPflash firefly luciferase reporter plasmid and 10 ng of a constitutive-active Renilla luciferase plasmid using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer’s instructions. Luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (E1960, Promega). Firefly luciferase values were normalized to Renilla luciferase control values.
For Smad and TCF reporter assays, we used the Smad reporter, TCF reporter and the Negative Control from the Cignal 45-Pathway Reporter Array (Qiagen, Lenti Reporter Assays). This assay is based on dual-luciferase technology and Negative control serves as a specificity control. The Smad reporter consists of Smad2/3/4 transcription factors-responsive firefly luciferase construct and a constitutively expressing Renilla luciferase construct. Cells were transduced with lentiviral particles for assessing canonical TGFβ activity, which is determined by comparing the normalized luciferase activities of the reporter in treated versus untreated cells.

The Cignal 45-Pathway–Reporter Array (Qiagen, Toronto, ON, Canada) [54 ] was used to measure the relative activity of multiple signaling pathways in B16-BL6 cells treated with 100 µg/mL of the ethanol extract of Uncaria tomentosa. In brief, the cells were transfected with two plasmids, a firefly luciferase plasmid linked to pathway-specific promoter elements and a constitutively expressing Renilla luciferase plasmid, using Attractine transfection reagent. The cells were treated with the Uncaria extract or control media for 24 h, and the amount of the two luciferase reporters was measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). The relative level of luciferase was determined by subtracting the luminescence of the negative control plasmid and dividing by the Renilla luminescence level in each sample. The effect of Uncaria tomentosa treatment was determined by dividing the luminescence for the Uncaria-treated samples by the media-treated control samples for 2 independent experiments.

Forty-five transcription factors’ activities were examined by Cignal 45-Pathway Reporter Array (Qiagen). Cells were planted in the 96-well plates provided in this kit, following the transfection, treatment of indicated drugs and luciferase measurement according to the protocol of this kit.

To identify the signalling pathways activated in chemoresistant cells, HCT/P and chemoresistant (HCT/FuR and HCT/OxaR) cells were analysed by using the Cignal 45‐Pathway Reporter Array (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions.