Fig. 5. Role of Dhx36, hnRNPK or Tial1 in the modulation of αENaC mRNA expression in alveolar epithelial cells. (A) Alveolar epithelial cells were transfected with expression vector encoding for Dhx36, hnRNPK or Tial1 RBPs. αENaC mRNA expression was quantified by RT-qPCR 72 h posttransfection and expressed as percentage ± SEM compared to cells transfected with an empty vector (pcDNA3) after normalization with β-actin. αENaC mRNA expression was significantly decreased by each RBP. *P<0.05 by one-sample t-tests compared to 100; n=6 for each experimental condition. (B) Alveolar epithelial cells were transfected with αENaC-Luc construct where a 3-kb portion of the αENaC promoter drives the expression of the Luc reporter gene in cells that were cotransfected with an expression vector coding for Dhx36, hnRNPK or Tial1 RBPs and the pRL-SV-40 coding for Renilla reniformis luciferase (RL) for normalization of the LUC signal. Luc and Renilla signals were measured 72 h posttransfection by Dual-Luciferase Reporter Assay (Promega Corporation) in an EnVision Multilabel reader. αENaC promoter activity was expressed as percentages ± SEM of Luc activity compared to cells transfected with an empty vector (pcDNA3) after normalization with RL. Overexpression of hnRNPK significantly inhibited αENaC promoter activity, whereas overexpression of Dhx36 and Tial1 had no effect. *P<0.05 by Kruskal-Wallis test and Dunn's post-hoc test compared to empty vector; n≥3 from different animals were tested in duplicate for each experimental condition. (C) Alveolar epithelial cells were cotransfected with the pTRE-tight plasmid encoding V5-αENaC mRNA along with an expression vector for Dhx36, hnRNPK or Tial1 RBPs and the pTet-Off plasmid. V5-αENaC mRNA expression was quantified by RT-qPCR 72 h posttransfection and expressed as percentage ± SEM of V5-αENaC mRNA compared to cells transfected with an empty vector (pcDNA3) after normalization with tTA-Ad. Overexpression of Dhx36 and Tial1 significantly inhibited V5-αENaC mRNA expression, whereas overexpression of hnRNPK had no effect. *P<0.05 by the Kruskal-Wallis tests and Dunn's post-hoc test compared to empty vector; n≥3 from different animals were tested in duplicate for each experimental condition. (D) Endogenous αENaC mRNA expression estimated by RT-qPCR 72 h after transfection by electroporation of alveolar epithelial cells with 500 nM siRNA against Dhx36 or Tial1. αENaC, Dhx36 and Tial1 mRNA expression ± SEM were normalized with the expression level of alveolar epithelial cells from the same series transfected with a control siRNA (siCtrl). Inhibition of Dhx36 or Tial1 had no effect on αENaC mRNA expression. *P<0.05 by Mann-Whitney U-tests between Ctrl and siRNA treated cells; n≥3 for each experimental condition. Representative immunoblots of Dhx36 and Tial1 knockdown are presented.