Silencer siRNA Knockdown of Transcription Factors

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.