Genmute transfection reagent

BEAS-2B cells were obtained from ATCC (Manassas, VA) and cultured in Hite’s Medium (DMEM F12 with 10% fetal bovine serum, 2 mM l-glutamine, 1x non-essential amino acids, 10 mM HEPES, 1 mM sodium pyruvate, and 1x Penicillin Streptomycin) in 10 cm dishes. Cells were transfected with 20 nM dicer substrate small interfering RNA (dsi RNA, Integrated DNA Technologies, Coralville, Iowa) using GenMute transfection reagent (Signagen, Frederick, MD) once 60–70% confluent. 48 hrs later lysate was collected and processed for immunoblotting.

TOLLIP gene in primary HBE and BEAS-2B cells was knocked down using Stealth siRNA duplex oligoribonucleotides specific for human TOLLIP (5’-CCAAGAAUUACGGCAUGACCCGCAU-3’; 5’-AUGCGGGUCAUGCCGUAAUUCUUGG-3’) obtained from Invitrogen. Scramble siRNA oligonucleotides were used as controls. Transfection of siRNA was performed using GenMute transfection reagent (SignaGen Laboratories). For transient overexpression of TOLLIP in BEAS-2B and primary HBE cells, TOLLIP-V5 plasmid was transfected into these cells using Lipofectamine 2000 (Invitrogen). To induce TOLLIP expression in the stable inducible BEAS-2B cells, the cells were treated with doxycycline (Sigma) at 2 μg/ml for 48 hours before experiment. Both overexpression and knocking down of TOLLIP protein in treated cells were confirmed by immunoblotting.

Transient miR-181a knockdown or overexpression was achieved with syn-hsa-miR-181a miScript miRNA mimic, control miR, anti-hsa-miR-181a miScript miRNA inhibitor, and miScript inhibitor negative control (Applied Biosystems). Transfections were performed with GenMute transfection reagent (SignaGen Laboratories, Gaithersburg, MD). pmiRZIP lentivector expressing anti-miR-181a or control sequence (SBI, Mountain View, CA) was used for permanent miR-181a knockdown experiments. Transduction-ready FIV-based pseudoviral particles were generated using pPACK-H1 Lentivector Packaging System together with 293TN cell line (SBI), at a titer of 1.06 × 10⁹ IFU/ml. Control was Lenti-scramble Hairpin control pseudoviral particles at a titer of 1 × 10⁹ IFU/ml. Cells were cultured in 12-well plates at a density of 1× 10⁵/well for 1 day, infected by pseudoviral particles (using a multiplicity of infection of 100 viruses per cell) and cultured for 72 hrs, then selected for puromycin (5µg/ml) resistance for stable cell lines. Stably transduced cells were used for vitro and in vivo experiments. Cells were transfected with human regulator of G-protein signaling 16 cDNA (RGS16, NM_002928) cloned into a pCMV vector (pReceiver-M02) and negative control Vector (EX-NEG-M02) (GeneCopoeia, Rockville, MD) and selected for neomycin resistance.