Tri reagent system

CI-huThyrEC human thyroid epithelial cells were purchased from InSCREENeX (cat. no. INS-CI-1017; Braunschweig, Germany). Primary human thyroid fibroblasts were purchased from ScienCell Research Laboratories (cat. no. 3730; Carlsbad, CA, USA). All the cells were grown according to suppliers’ instructions using the recommended media and other reagents (huThyrEC medium, cat. no. INS-ME-1017; InSCREENeX), epithelial cell coating solution (cat. no. INS-SU-1020; InSCREENeX), and fibroblast medium (cat. no. 2301; ScienCell Research Laboratories). Human recombinant TNF was purchased from PeproTech, Inc. (Cranberry, NJ, USA) and used at 25 ng/mL for the experiments. Human recombinant TGF-β1 was obtained from PeproTech and used at 10 ng/mL. In one set-up, thyroid epithelial cells were also cultured on depleted medium which consisted of huThyrEC basal medium with 1% fetal bovine serum, amphotericin B, penicillin, and streptomycin. The cells were seeded onto six-well plates and cultured until they reached a sufficient confluence. Medium containing TNF or TGF-β1 was added, and after the duration of the experiment the cells were lysed with the Tri reagent system (Sigma-Aldrich Corp., St. Louis, MO, USA). The experiments were repeated three times (N = 3) with two technical replicates per experiment, using the average of the two as the final value for the experiment.

Total RNA was isolated using the TRI-reagent system (Sigma) from 5 × 10⁵ cells. To quantify the amount of TF mRNA, real-time RT-PCR was carried out in triplicates using primer sets designed to detect TF and β-actin and the absolute amounts of TF mRNA quantified as previously described [35 (link)]. Single-step RT-PCR was carried out in triplicates using 100 ng of total RNA from each sample tested. A set of previously prepared standard TF mRNA ranging 0.05-10 ng was included [35 (link)]. To assess the expression of PAR2, real-time RT-PCR was carried out in triplicates using 100 ng of total RNA from each sample using primer sets designed to PAR2 and β-actin mRNA. After the amplification, the ratios of PAR2 mRNA were determined with respect to that present in MIA-PaCa-2 cells which express low levels of PAR2, using the 2^-ΔΔCT method [36 (link)]. The reaction was carried out at an annealing temperature of 60 °C using the GoTaq® 1-Step RT-qPCR System (Promega Corporation Ltd, Southampton, UK) on an iCycler thermal cycler (Bio-Rad, Hemel Hempstead, UK) and the data analysed. The primers used were:

TF-forward: 5'-TACAGACAGCCCGGTAGAGTG-3',

TF-reverse: 5'-GAGTTCTCCTTCCAGCTCTGC-3',

PAR2-forward: 5'-GAGCCATGTCTATGCCCTGT-3'

PAR2-reverse: 5'-GACACTTCGGCAAAGGAGAG-3'

β-actin-forward: 5'-TGATGGTGGGCATGGGTCAGA-3',

β-actin-reverse: 5'-GTCGTCCCAGTTGGTGACGAT-3'

Total RNA was extracted from cells using the Tri‐Reagent system (Sigma‐Aldrich, Dorset, UK). Concentration was determined spectrophotometrically at OD260 on a Nanodrop spectrophotometer (Thermo Scientific, Hemel Hempstead, UK). In a 20‐μL volume, 1 μg of total RNA was incubated with 10× RT buffer, 100 mmol/L dNTP mix, 10× RT random primers, 50 U/μL MultiScribe reverse transcriptase, and 20 U/μL RNase inhibitor. The reverse transcription was carried out at 25°C for 10 min, 37°C for 120 min, and then the reaction was terminated by heating to 85°C for 5 min.