2.5. RT-qPCR

The TRIzol LS reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the cells’ total RNA following the product’s standard protocol. The RNA concentration was determined with Nanodrop 2000 spectrophotometer, and its integrity was verified by electrophoresis with 1.5% agarose gel in Tris-Borate buffer using GreenSafe (NZYTech, Lisboa, PT, Portugal). Complementary DNA (cDNA) from 1 µg of RNA was obtained using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, 28025013, Thermo Fisher Scientific) following the provider’s standard protocol. The 18S ribosomal RNA (rRNA) was used as the housekeeping gene. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) experiments were performed with the FastStart DNA Master SYBR Green I kit (Roche, Basel, Switzerland) in a LightCycler 2.0 instrument (Roche) to gene amplification. The used primers were TJP1 (tight junction protein 1): FW-5′-gccattcccgaaggagttga-3′, RV-5′-atcacagtgtggtaagcg-3′; CDH2 (cadherin-2): FW-5′-ctggagacattggggacttc-3′, RV-5′-gagccactgccttcatagt-3′; and VIM (vimentin): FW-5′-ggaccagctaaccaacgaca-3′, RV-5′-aaggtcaagacgtgccagag-3′. rRNA18S [FW-5′-agtgaaactgcgaatggctc-3′, RV-5′-ctgaccgggttggttttgat-3′]. Relative expression was quantified by the comparative 2^∆∆Ct method [49].