Beas 2b

Normal human lung epithelial cells BEAS-2B (iCell-h023) and NSCLC cell lines A549 (iCell-h011), NCI–H1299 (iCell-h153), NCI–H1650 (iCell-h155-001b), and NCI–H1975 (iCell-h156) were obtained from iCell (Shanghai, China). They were cultivated in Dulbecco's modification of Eagle's medium Dulbecco (DMEM) (iCell-0001), F–12K (iCell-0007), or RPMI-1640 (iCell-0002) media supplemented with 10% phosphate buffer saline (PBS) and 100 U/mL penicillin-streptomycin.

Human bronchial epithelial cell line BEAS-2B was purchased from iCell Bioscience, Inc. (Shanghai, China) and stained for Pan Cytokeratin (PCK) for cell identification. Briefly, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 10 min at room temperature. The cells were washed with PBS and then incubated with primary antibody for PCK (Cat# bs- 5352r, 1:100 dilution; Bioss, Woburn, MA, USA) overnight at 4°C, washed with PBS and incubated with secondary antibody cy3 (Cat# AS007, 1:200 dilution; ABclonal, Woburn, MA, USA) for 1 h at room temperature. For negative control, the cells were incubated with PBS instead of anti-PCK antibody. The nuclei were stained with 1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MA, USA) in the dark for 1 min at room temperature, and the slices were sealed with blocking solution containing anti-fluorescence quencher, and observed under fluorescence microscope (CKX53; Olympus, Tokyo, Japan) equipped with a Zyla 4.6 sCMOS camera to record the images. Immunolabeling was assessed by ImageJ software analysis program (NIH Image, Bethesda, MD, USA).

Normal lung epithelial cell line BEAS-2B and four NSCLC cell lines, namely, A549, H23, H1299, and H520 were purchased from iCell (Shanghai, China). All the five cell lines were incubated in a 5% CO₂ humidified incubator at 37 °C. The total RNA of all cell lines was isolated using Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was reversely transcribed from the total RNA by using a reverse transcription kit (Promega) and PrimeScript RT Master Mix (Takara, Japan). The target gene was measured with SYBR Green assay in the 96-well plates following the manufacturer’s instructions (Applied Biosystems, Carlsbad, CA, USA). The primers used were as follows: MRPL13, forward: 5′-ACATAAACCTGTGTACCATGCAC-3′; MRPL13, reverse: 5′- GGTAGCCAGTATGCGAAGAGT-3′; GAPDH, forward: 5′-AC CACAGTCCATGCCATCAC-3′; and GAPDH, reverse: 5′-TCCACCACCCTG TTGCTGTA-3′.

The human lung cancer cell lines A549, H1975, H1299 and the human bronchial epithelial cell line BEAS-2B (iCell Bioscience Inc, China) were grown in a dampish gaseous environment with 5% CO₂ at 37 °C. The A549, H1299 and H1975 cells were cultured with RPMI 1640 medium (Gibco, Invitrogen, USA), and the BEAS-2B cells were cultured with DMEM medium (Gibco, Invitrogen, USA). The RPMI 1640 and DMEM medium were complemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, USA) and 1% penicillin-streptomycin solutions (TIANHANG Biotech Company, China) [30 (link)].