H1975

Independent shRNAs targeting PAQR4 were constructed using a pLKO.1 vector. The two independent PAQR4 targeting sequences are: shRNA#1, 5'-GCAGGCTCCGTGCTCTATCAC-3'; shRNA#2, 5'-CGTCTTGCTCTGAGAGTTCAA-3'. The pNFE2L2 (NRF2)-ENTER (Gene ID: NM_006164) and pKEAP1-ENTER (Gene ID: NM_203500) plasmids were purchased from Vigene Biosciences Inc., and were sub-cloned into pCDNA3.1. Nrf2 was N-terminal tagged with 6×Myc, and Keap1 was N-terminal tagged with 3×HA. The full length cDNA of PAQR4 (Gene ID: NM_152341.5) was synthesized by Shanghai Generay Biotech, and sub-cloned into pCDH-MSCV-E2F-eGFP lentiviral vector or pCDNA3.1 vector with a 3×Flag at the C-terminus. The lentiviruses were generated according to the manufacturer's protocol, stable cell lines were generated by lenti-viral infection. The BEAS-2B cell line was a gift from Dr. Hongbin Ji at SIBS, CAS, China. HEK-293T was obtained from ATCC. A549, H1299, H1975, H1650 were purchased from Cobioer, China with STR document, BEAS-2B, A549, H1650, H1975, H358, GLC-82 and SPC-A1 cells were cultured in RPMI1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. H1299 and HEK-293T cells were cultured in DMEM medium (Corning).

The BEAS-2B cell line was purchased from the Cell Bank of theKunming Institute of Zoology and was cultured in BEGM media (Lonza, CC-3170). Lung cancer cell lines, namely, h1650, A549, SPC-A1, and H1975, were purchased from Cobioer, China with STR documents, and were cultured in RPMI-1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

The BEAS-2B cell line was purchased from cell bank of Kunming Institute of Zoology, and cultured in BEGM media (Lonza, CC-3170). Lung cancer cell lines, including A549, H1650 and H1975 were purchased from Cobioer, China with STR document, A549, H1650 and H1975 cells were all cultured in RPMI1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

The BEAS-2B cell line was purchased from the Cell Bank of Kunming Institute of Zoology and cultured in BEGM media (CC-3170; Lonza, Basel, Switzerland). Lung cancer cell lines, including A549, H1299, H358, HCC827, SPC-A1, H1650, and H1975, were purchased from Cobioer (Nanjing, China), with STR document, and were cultured in RPMI-1640 medium (Corning, Corning, NY, United States) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The normal control (NC), miR-125b-5p mimics, and miR-125b-5p inhibitors were purchased from RiboBio (Guangzhou, China). Cells were transfected with the indicated miRNA mimics or NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) and then collected for various experiments.

Cell culture, RNA isolation, and real-time polymerase chain reaction (PCR) assay were performed as published (Shi et al., 2019 (link)). The BEAS-2B cell line was purchased from the cell bank of Kunming Institute of Zoology and cultured in BEGM media (Lonza, CC-3170). HEK-293T was obtained from ATCC. Lung cancer cell lines, including A549, H1299, and H1975, were purchased from Cobioer (China) with STR documents; A549, H1299, and H1975 cells were all cultured in RPMI 1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HEK-293T cells were cultured in a Dulbecco modified eagle medium (Corning). The detail information of primer used in this study are as follows: NCAPG-F: GAG​GCT​GCT​GTC​GAT​TAA​GGA, NCAPG-R: AAC​TGT​CTT​ATC​ATC​CAT​CGT​GC, TYMSOS-F: ATG​ACG​CCC​GCC​TCG​GGG​GCC, TYMSOS-R: TCA​GGA​AGG​ACG​ACC​GCA​CGG​GCA​CC, FOXM1-F: CGT​CGG​CCA​CTG​ATT​CTC​AAA, FOXM1-R: GGC​AGG​GGA​TCT​CTT​AGG​TTC, miRNA-214-3p-F TTT​TTA​CTA​CTA​TGG​CGG​GTG​ATA​AAA​CGT​GTA, miRNA-214-3p-R: GCA​AGC​TGT​AAT​CGA​CGG​GAA​GAG​CAT​GCC​CAT​CC, ACTIN-F: CTTCGCGGGCGACGAT, and ACTIN-R: CCA​TAG​GAA​TCC​TTC​TGA​CC. The expression quantification was obtained with the 2^−ΔΔCt method.

Independent shRNAs against different genes targeting different regions were constructed using a pLKO.1 vector. The 3XFlag C-terminal tagged forms of different overexpression genes were synthesized and cloned into a pCDH-MSCV-E2F-eGFP lenti-viral vector. All of the constructs were verified by sequencing, detailed cloning information can be provided upon request. The lenti-viruses were generated according to the manufacturer’s protocol. Briefly, supernatants containing different lenti-viruses generated from HEK-293T cells were collected 48 and 72 h post-transfection. HEK-293T was purchased from ATCC, BEAS-2B was kindly provided by Dr. Hongbin Ji at SIBS, CAS. H1975, A549, NCI-H838, H1299. and NCI-H1650 were purchased from Cobioer, China with STR document, GLC-82, SPC-A1 were gifts from Dr. Yunchao Huang, and A549-DDP was a gift from Dr. Shiyong Sun at Emory University, Atlanta, USA. All cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin then incubated in a humidified atmosphere with 5% CO₂ at 37 °C. Cisplatin was purchased from Sigma (Cat#p4394, USA).