Silencer sirna transfection kit

Knockdowns of Cx43 were obtained by transfection of B16F10 cells with esiRNA against Cx43 (siCx43; Mission, Sigma-Aldrich, St. Louis, MO, USA), a pool of independent target-specific siRNAs against Cx43. A siRNA of scrambled sequence (siRNA Scrambled; Ambion, Thermo Fisher Scientific, Austin, TX, USA; siScr) without target in the murine genome was used as a negative control. Transfections were performed using the Silencer siRNA Transfection Kit (Ambion, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, a transfection complex was generated mixing the siPORT amine transfection agent with the transfection medium OPTIMEM™ (Gibco). Then, the transfection complex was incubated for 10 min with 30 nM of siCx43 or siScr. B16F10 cells were incubated with siRNA-containing transfection complex for 6 h. After this period, the transfection complex was replaced with complete cell-culture medium. After 72 h, cells were harvested and used in different experiments.

Lentivirus carrying shRNA sequence to the target gene was prepared using a three-plasmid transfection method [36 (link)] and used to infect cells. Cells with gene knockdown (SAE2^KD, EGFR^KD, FAK^KD, c-Met^KD or nAchRα7^KD) were selected using 1 μg/mL puromycin. The siRNA used for silencing gene expression was obtained from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Centre, Academia Sinica, Taipei, Taiwan). Knockdown of cirRNA CCDC66 (cirRNA CCDC66^KD) was carried out by direct delivery of siRNAs (siJCT1 and siJCT2) [24 (link)] into H23 and H1975 cells using Ambion Silencer® siRNA transfection kit [10 (link)]. Reduction of cirRNA CCDC66 was determined by RT-PCR and gel electrophoresis.

Silencer siRNA Transfection Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for the knockdown of selected genes (NF-κB, c-Myc and STAT3), according to the manufacturer’s instructions (Figure S1). The transcription factors were selected according to the literature, as the factors involved in the process of airway remodeling, but also as suggested elements of the relaxin pathway in this process [23 (link),101 (link),102 (link),103 (link),104 (link),105 (link)]. The cells were treated with 20 nM siRNA mixture against NF-κB (NCBI accession no. NM_001145138.1), c-Myc (NCBI accession no. NM_002467.4) and STAT3 mRNA (NCBI accession no. NM_003150.3), (Thermo Fisher Scientific, Waltham, MA USA) for 48 h. The same concentration of scrambled siRNAs was used as the negative control. The knockdown efficiency was evaluated after 48 h of transfection. The measurement of gene knockdown was analyzed according to the manufacturer’s protocol by qPCR. The data presented are normalized to the samples treated with control siRNA.