Qrt supermix

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The reverse transcription of 1 μg RNA was carried out using qRT SuperMix (Vazyme, Nanjing, China). A real-time-polymerase chain reaction (RT-PCR) analysis was carried out using the ABI Stepone Plus system with the AceQ SYBR Green Master Mix (Vazyme, Nanjing, China). The primers were synthesized by Bioneer, and the primer sequences for the human genes were: VE-cadherin, 5′-CCAGAATTTGCCCAGCCTA-3′ and 5′-TTACTGGCACCACGTCCTTGTC-3′; Vimentin, 5′-TGCCGTTGAAGCTGCTAACTA-3′ and 5′-CCAG-AGGGAGTGAATCCAGATTA-3′; Nrf2, 5′-TTCCCGGTCACAT-CGAGAG-3′ and 5′-TCCTGTTGCATACCGTCTAAATC-3′; Smad7, 5′-GGACAGCTCAATTCGGACAAC-3′ and 5′-GTACACCCACACACCATCCAC-3′; β-actin, 5′-AAGACCTGTACGCCAACACAGT-3′ and 5′-AGAAGCATTTGCGGTGGACGAT-3′. Amplified expressions of gene transcriptions were normalized to β-actin expression. Cycle threshold (ΔΔCt) values were calculated for each experimental group, which indicated the number of available template cDNA in each reaction. The gene expression levels were compared using values of 2^−ΔΔCt.

The total RNA in the lung tissue was harvested using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using qRT SuperMix (Vazyme, JiangSu, China). RT-qPCR was performed with AceQ SYBR-Green Master Mix (Vazyme). The following primers were used for PCR analysis: ICAM-1 forward, 5′-GTGATGCTCAGGTATCCATCCA-3′ and reverse, 5′-CACAGTTCTCAAAGCACAGCG-3′; VCAM-1 forward, 5′-TTGGGAGCCTCAACGGTACT-3′ and reverse, 5′-GCAATCGTTTTGTATTCAGGGGA-3′; TNF-α forward, 5′-GGAACACGTCGTGGGATAATG-3′ and reverse, 5′-GGCAGACTTTGGATGCTTCTT-3′; IL-8 forward, 5′-AGCAGTCCCAACTAAGCAGTA-3′ and reverse, 5′-CAGCCAGTAGAGGATGCTGA-3′; and β-actin forward, 5′-GAGTCCTACGACATCATCGCT-3′ and reverse, 5′-CGTCCGACATAGTTTGGGAAA-3′. The cycle threshold (CT) method was used to analyze the data in each experimental group (19 (link)).