RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Cells were pretreated with or without MaR 1 (10 nM) for 30 min, followed by the addition of TGF-β1 (10 ng/ml) for 24 h. Total cellular RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and 2 µg RNA was reverse transcribed into cDNA using the RevertAid^™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The specific primer sequences (Invitrogen; Thermo Fisher Scientific, Inc.) used were as follows (5′-3′): α-SMA forward, GAC AAT GGC TCT GGG CTC TGT AA and reverse, ATG CCA TGT TCT ATC GGG TAC TT; collagen α-1(I) chain (COL1A1) forward, GAG GGC CAA GAC GAA GAC ATC and reverse, CAG ATC ACG TCA TCG CAC AAC; and GAPDH forward, AGT GCC AGC CTC GTC TCA TAG and reverse, CGT TGA ACT TGC CGT GGG TAG. Each specific gene product was amplified by real-time PCR using SYBR-Green Master Mix in the StepOne™ Real-Time PCR system (Thermo Fisher Scientific, Inc.). PCR thermal cycling was conducted at 95°C for 15 sec, followed by denaturing at 95°C for 5 sec, annealing at 60°C for 60 sec and a final extension step at 72°C for 10 sec, for a total of 40 cycles. The reaction was duplicated for each sample. The relative expression levels of each transcript were normalized to GAPDH expression. The quantitative fold changes in gene expression were calculated using the 2^−ΔΔCq (18) method following normalization to GAPDH.