Total RNA was extracted from harvested cells (fresh and stimuli) using an RNase Plus Mini Kit (cat. no. 74104; Qiagen GmbH). Total RNA was reverse transcribed into cDNA using the PrimeScript II 1st Strand synthesis Kit (cat. no. 6210A; Takara Bio, Inc.) according to the manufacturer's protocol. Subsequently, qPCR was performed using the SYBR-Green method (Takara Bio, Inc.) and the Mx3000P qPCR System (Agilent Technologies, Inc.) as previously described (25-27). The sequences of the primers used for qPCR are listed in Table I. The expression levels of transcription factors, forkhead box P3 (FoxP3), T-box transcription factor TBX21 (Tbet), GATA binding protein 3 (GATA3) and retinoic acid receptor-related orphan receptor C (RORC), cytokines, including IL-4, IL-10, IL-17A, IFN-γ and TGF-β, and molecules and genes that have been modified by asbestos long-term exposure, including MMP-7, NNT and CXCR3, as determined in our previous studies (16-20,25-27). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 20 sec; and melting curve analysis at 95°C for 1 min, 60°C for 30 sec and 95°C for 30 sec. mRNA expression levels were quantified using the 2−∆∆Cq method (44)

Sequences of primers used for reverse transcription-quantitative PCR.

FoxP3, forkhead box P3; Tbet, T-box transcription factor TBX21; GATA3, GATA binding protein 3; RORC, retinoic acid receptor- related orphan receptor C; CXCR3, C-X-C motif chemokine receptor 3; MMP-7, matrix metalloproteinase-7; NNT, nicotinamide nucleotide transhydrogenase; F, forward; R, reverse.

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