Fig 2: Quantification of a-ENaC, ß-ENaC and ?-ENaC gene expression using reverse transcription-quantitative PCR. (A) a-ENaC, ß-ENaC and ?-ENaC mRNA expression levels were determined in the lung tissue of mice with LPS-induced lung injury following alcohol treatment (n=6 per group). (B) a-ENaC, ß-ENaC and ?-ENaC mRNA expression levels were determined in alveolar epithelial cells following ethanol treatment. Values were normalized to endogenous GAPDH expression levels and were presented as the relative expression levels. The data represent three independent experiments. Data are presented as the mean ± standard deviation. ?P<0.05 vs. control; #P<0.05 vs. LPS; *P<0.05 vs. LPS + ethanol; ##P<0.05 vs. ethanol. ENaC, epithelial sodium channel; LPS, lipopolysaccharide.

Fig 3: MiR-130b can be transferred into AT2 cells co-cultured with BMSCs and increases protein level a/?-ENaC in AT2 cells. a Transfected miR-130b labelled with Cy3 was localized in AT2 cells co-cultured with BMSCs for 24 h. BMSCs were transiently transfected with Cy3-miR-130b after which they were co-cultured with mouse AT2 cells and then fixed. Nuclei were counterstained by 4,6-diamidino-2-phenylindole (DAPI, blue; left panel), and the representative staining images were shown in CY3-miR-130b positive AT2 cells (red; middle panel). The merged images were seen (right panel). Scale bar, 10 µm. b The result of real-time PCR assays shows miR-130b level in AT2 cells treated with LPS for 12 h and/or co-cultured with BMSCs for 24 h. The relative level of miR-130b were calculated as miR-130b/U6 ratio. c Representative Western blot bands of a- and ?-ENaC protein expression in AT2 cells transfected with miR-130b mimic/inhibitor for 72 h. Blots for ß-actin were used as internal controls. d, e Graphical representation of data obtained from Western blot assays for a- and ?-ENaC subunits. Bands were quantified using gray analysis (a-ENaC/ß-actin and ?-ENaC/ß-actin). **P < 0.01, compared with control; &P < 0.05, compared with LPS; #P < 0.05, compared with miR-130b mimic negative control (NC); $P < 0.05, compared with miR-130b inhibitor negative control (Inhibitor NC), n = 4–6

Fig 4: Effects of CRO on the protein expression level of ENaC a- and ?-subunits in H441 cellsH441 cells were exposed to 80 µM CRO for 0 to 24 h and then proteins were extracted and analysed by western blot. (A, B) Western blots of a- and ?-ENaC protein demonstrating reductions in levels over time. Blots for ß-actin were used as internal controls. (C, D) Graphical representation of data obtained from three sets of western blot assays for which bands were quantified using gray analysis (a-ENaC/ß-actin and ?-ENaC/ß-actin). Data are shown as the mean ± SE, *P < 0.05, **P < 0.01, compared with control.

Fig 5: Characterization of pancreatic ductal epithelial cells. (a–f, l: Characterization of organoids). a Characterization of pancreatic ductal organoids using epithelial cell markers, b cytokeratin 19 (KRT 19, c E-cadherin, e sodium transport channel (ENaC), and f ZO-1. d Hematoxylin and eosin (H&E) image shows the orientation of pancreatic ductal epithelial cells in spheroid of the organoid. (g–k: Characterization of monolayer of pancreatic ductal epithelial cells (PDECs)). g Phase contrast and j H&E images show monolayer of PDECs formed from the organoids. Monolayer of PDECs showed positivity for tight junction h ZO-1, i F-actin and KRT 19, and k cystic fibrosis transmembrane conductance regulator (CFTR). l RNA-sequencing data was obtained from the pancreatic ductal organoids and verified the PDEC origin (n = 4 sample preparation from the same patient). Data are mean ± SD. Scale bars: 10 µm (k), 20 µm (b, c, e, f, h–j enlarge), 50 µm (d, g, j), and 500 µm (a)