Pcdna3.1 plasmid

The human alveolar epithelial A549 cell line was used to analyze the effect of miR142 overexpression on CD209L levels. Cells were cultured in DMEM supplemented with 10% FBS (both from Fisher Scientific) and 100 U/mL penicillin-streptomycin (GE Healthcare Life Sciences, Piscataway, NJ, USA). MiR142 was cloned in pcDNA3.1 plasmid (Addgene, Watertown, MA, USA). DH5α E. coli (Thermo Fisher, Waltham, MA, USA) was transformed with control pcDNA3.1 and miR142 plasmids, followed by plasmid DNA isolation using an EasyPrep kit (Bioland Scientific, Paramount, CA, USA). A549 cells were transfected with 2.5 µg plasmid DNA using Polyethyleneimine (PEI) reagent (Polysciences Inc., Warrington, PA, USA) for 24 h. MiR142 overexpression was confirmed by RT-PCR (Table 1) as described above.

In order to overexpress each of the candidate microRNA selected, the pre‐microRNA sequence of miR‐431 or miR‐636 were retrieved from miRBase (http://www.mirbase.org/), amplified by PCR and inserted into pcDNA 3.1 plasmid (Addgene) downstream to the promoter by standard PCR/restriction enzyme‐based cloning methods. The insert sequence for the pre‐microRNAs is presented in Figure S2. The constructs thus generated were validated by their transfection in HeLa cells that revealed a 3‐ to 14‐fold increase in miR‐636 and miR‐431 levels, respectively (data not shown). An AAV plasmid containing either an inducible caspase 9 gene (iCasp9) or an enhanced green fluorescent protein (EGFP) as transgene45, 46 was utilized for packaging in the presence and absence of miR‐431 and miR‐636 constructs.

The pcDNA3.1 plasmid expressing wild-type HIF-1α was obtained from Addgene (Cambridge, MA, USA). Transfection into H1299 cells was performed using TurbofectTM transfection reagent (Thermo Scientific, Waltham, MA, USA).

A549 and H1975 cells were planted in six-well plate 24 h before transfection. When they were about 70% confluent, cells were transfected with siRNA targeting specific genes or negative control (RealGene, Nanjing, China) by using the Lipofectamine RNAimax reagent (Invitrogen, USA) according to the protocol provided by the manufacturer. The siRNA sequences for linc00630 were 5′-CAGAAGGAGUGAACUGCUAAG-3′ (Sense) and 5′-UAGCAGUAGACUGGUUCUGGG-3′ (Antisense) for 204 site and 5′-GCAAGCCUGCAUGGUACAUTT -3′ (Sense) and 5′-AUGUACCAUGCAGG CUUGCTT -3′ (Antisense) for 1021 site. pCDNA3.1 plasmid was from Addgene (Cambridge, USA). The pCDNA3.1-Puro vector for LINC00630 overexpression was from Applied Biological Materials (ABM) Incorporation. The transfection reagent PowerFect™ In Vitro siRNA was from SignaGen Laboratories (Rockville, USA). All experiments were performed after 48 h of transfection. NSCLC cells tranfected with Vector or p-linc00630 plasmid were screened by puromycin (4 μg/ml) for higher expression efficiency.

Normal esophageal squamous (NES) cells (gift from R. Souza, University of Texas Southwestern Medical Center, Dallas, TX) were cultured in a supplemented 3:1 mixture of Dulbecco’s modified Eagle’s medium/Ham's F12 medium (Invitrogen, Carlsbad, CA) as previously described.³⁰ (link) Cell numbers were determined by trypan blue cell exclusion method after 48-hour transfection.
Plasmid sequence (Supplementary Figure 1) and transfection miRNA expression plasmids were cloned by replacing the insert from a pcDNA3.1 plasmid (plasmid #21114; Addgene, Cambridge, MA) with inserts cloned by PCR (primers detailed in Supplementary Table 1). Plasmid cloning was validated by Sanger sequencing using a CMV-F primer (Supplementary Figure 1). Transfection was performed by using Lipofectamine 3000 with expression plasmid or anti-miR (miRIDIAN microRNA Hairpin Inhibitor; Dharmacon, Lafayette, CO) according to the manufacturer's protocol. All data reflect at least 2 biological replicates and refer to fold change versus empty vector, with each experiment normalized to untransfected controls, 48 hours after transfection, unless otherwise stated.