H1299

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).

Lung adenocarcinoma cells A549 and H1299 were obtained from Shanghai Institutes for Biological Sciences (SIBS) and authenticated by STR profiling. 10% fetal bovine serum (Gibco, USA) was added to RPMI-1640 medium (Corning, USA) for incubation of A549 and H1299 cells and added to DMEM medium (Corning, USA) for incubation of Beas2B and HEK293 cells.

Two human NSCLC cell lines H1299 and SK-MES-1 were bought from the cell banks of the Shanghai Institutes of Biological Sciences. All cell lines were tested and authenticated before use. H1299 cells were cultured in RPMI-1640 medium (Corning) containing 10% fetal bovine serum (FBS). SK-MES-1 cells were cultured in minimum essential medium (Gibco) containing 10% FBS.

The human NSCLC cell line H1299 was purchased from Chinese Academy of Sciences Cell Bank (Beijing, China). These cells were authenticated by short tandem repeat method. The H1299 cells were cultured in RPMI-1640 medium (CORNING, 10–040-CVR, USA), which contained 10% fetal bovine serum (GIBCO, USA) and 1% penicillin/streptomycin (Shanghai, China) in a 5% CO₂ incubator (3111, Thermo) at 37 °C. H1299 cells were treated with 1 × 10^–6 mol/L of vitamin D (D1530, Sigma) for 96 h.

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.

Human NSCLC A549 cells were purchased from ATCC (USA) and H358 (KCLB No. 25807), H1299 (KCLB No. 25803), and human lung fibroblast IMR90 cells from Korean Cell Line Bank (KCLB No. 10186). A549, H358, and H1299 cells were maintained in DMEM and IMR90 cells in Minimum Essential Medium (MEM; Corning, USA) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). All cells were maintained at 37˚C in a humidified atmosphere of 5% CO₂. The use of human NSCLC patient’s tissue samples for this study was approved by the International Review Board of Eulji University (EU 18–14). The NSCLC tissues were originated from human lung adenocarcinoma (10), bronchoalveolar carcinoma (3), squamous cell carcinoma (2), and large cell carcinoma (2). The cancer tissues of lung cancer patients used were obtained in pairs with normal lung tissues, of which eight normal lung tissues were pooled and used as a normal group in the qRT-PCR experiment.