Lipofectamine 2000 agent

TCF21 overexpression and knockdown was performed based on a modified protocol described previously [31 (link)]. Briefly, to establish the TCF21-expressing cell line, the cDNA encoding TCF21 (OriGene #SC12508) was cloned into the pWPI lentiviral backbone (#12254, Addgene, Inc. Cambridge, MA) to construct the transfer plasmid. Oligonucleotides encoding shTCF21 [31 (link)] and shDNMT1 [32 (link)] were inserted to the same plasmid. HEK293T cells (1 × 10⁶) were seeded into tissue culture dish until 50% confluence. Transfection was done in DMEM medium, co-transfecting with packaging vectors and Mission Lentiviral packaging mix (Sigma-Aldrich) and Lipofectamine 2000 agent (Invitrogen). Virons that produced at 24 h-incubation were purified with centrifugation at 3000 rpm and filtration through a 0.45 μm-pore-size membrane. These virons were used to transfect cells cultured in 6-well plates. Cells stably expressing TCF21, shTCF21, or shDNMT1 were selected at 48 h after transfection using 2 μg/mL puromycin.

To examine the regulation between lncRNA MDFIC-7 and miR-525-5p, U-CH1 or U-CH2 cells were co-transfected with pmirGLO-MDFIC-7-WT, which contains the predicted miR-525-5p binding site, or pmirGLO-MDFIC-7-mut, together with miR-525-5p mimic, inhibitor or mock. To investigate the effect of lncRNA MDFIC-7 and miR-525-5p on ARF6, cells were co-transfected with pmirGLO-ARF6-3′UTR-WT and pmirGLO-ARF6-3′UTR-mut, together with miR-525-5p mimic or pcDNA-MDFIC-7. Transfection was performed using Lipofectamine 2000 agent (Invitrogen). After transfection for 48 h, the luciferase activity was calculated using the Dual-Luciferase Assay Kit (Promega) on a GloMax 20/20 Luminometer. Firefly luminescence was normalized to the Renilla luminescence according to the manufacturer’s instructions.

Human LAD cell lines (A549, SPC-A1, H1299 and PC-9) and normal lung epithelial cells BEAS-2B were bought from the Cell Bank of the Chinese Academy of Sciences and incubated with Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technology Inc., Carlsbad, CA), containing 10% fetal bovine serum (FBS) with 5% CO₂ at 37 °C in humid air. All cell lines were authenticated via STR profiling before using.
The suppression of LINC02418 expression was achieved by sh-LINC02418#1/2. Sh-LINC02418#1 and sh-LINC02418#2 were obtained from GenePharma (Shanghai, China). LINC02418 and KNL1 were overexpressed with pcDNA3.1 vectors (Invitrogen, Carlsbad, USA). MiR-4677-3p mimics were applied to elevate miR-4677-3p expression. MiR-4677-3p mimics and NC mimics were also bought from GenePharma (Shanghai, China). The transfection of above plasmids was conducted by use of Lipofectamine® 2000 agent (Invitrogen).