Sybr green supermix with rox

RNA was extracted from HPMECs by Trizol Reagent (15596026, Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The RNA expression of IL-1β, IL-6, and E-selectin was detected by RT-PCR with mRNA-specific primers (Sangon Biotech, CN). One microgram of RNA was reverse-transcribed into cDNA using the Reverse Transcription System (4374966, Thermo Fisher Scientific, USA). RT-PCR was performed with SYBR Green Supermix with ROX (A25742, Thermo Fisher Scientific, UK) using a PCR detection system (7500fast, Applied Biosystems, CA). Ct values were used to quantify the mRNA levels, and the Ct value of each target mRNA was standardized to the Ct value of β-actin using the 2^-ΔΔCt method. The primer pairs used are listed in Table 1.

Using the TRIzol Reagent (15596026; Thermo Fisher Scientific, Carlsbad, CA, USA), RNA was extracted from the lung tissues and BEAS-2B cells. To quantify the amount of mRNA, cDNA was generated from 1 μg of RNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Milan, Italy), with the final volume of 20 μL (K1622; Thermo Fisher Scientific). Next, qRT-PCR was performed using SYBR Green Supermix with ROX (A25742; Thermo Fisher Scientific, Hertfordshire, UK) and a PCR detection system (7500fast; Applied Biosystems, Foster City, CA, USA). The results were statistically analysed using the 2^−ΔΔCt method, with β-actin as an internal normalisation control. All primer sequences are shown in Table 1.Table 1

Sequences of the primers used for quantitative real-time PCR.

	Gene	Forward primer (5′–3′)	Reverse primer (5′–3′)
Mouse	β-Actin	GTGCTATGTTGCTCTAGACTTCG	ATGCCACAGGATTCCATACC
	IL-6	TCTGCTCTGGAGCCCACCAAG	CCAGCATCAGTCCCAAGAAGGC
	TNF-α	AAGGGAGAGTGGTCAGGTTGCC	TGTGAGGAAGGCTGTGCATTGC
	IL-1β	GCAGCAGCACATCAACAAGAGC	AGGTCCACGGGAAAGACACAGG
BEAS-2B cell	β-Actin	CACGATGGAGGGGCCGGACTCATC	TAAAGACCTCTATGCCAACACAGT
	Nrf2	TCCAAGTCCAGAAGCCAAACTGAC	GGAGAGGATGCTGCTGAAGGAATC
	IL-6	CTGCAAGAGACTTCCATCCAG	AGTGGTATAGACAGGTCTGTTGG
	IL-1β	GAAATGCCACCTTTTGACAGTG	TGGATGCTCTCATCAGGACAG
	TNF-α	CAGGCGGTGCCTATGTCTC	CGATCACCCCGAAGTTCAGTAG