Fig 2: CFTR is aberrantly expressed in acinar cells of CFTRtm1HGU mice, but exocrine pancreatic function is maintained. (A) A transgenic mouse model containing a neomycin cassette and a part of exon 10 of the CFTR gene was used that has residual CFTR expression. (B) Immunofluorescence labelling of CFTR (Clone: M3A7, DLN-06996, Dianova) in wild-type and CFTRtm1HGU mice showed a clear localization of CFTR on the cellular membrane in wild-types and a prominent labelling in the cytoplasm of CFTRtm1HGU mice. A second antibody (Clone: A-3, Santa Cruz Biotechnology) was used for immunohistochemistry to verify the labelling. We confirmed the localization of CFTR at the cell membrane in wild-type mice, which was absent in CFTRtm1HGU animals. (C) Comparable stool chymotrypsin activities wild-type and CFTRtm1HGU mice indicate that exocrine pancreatic enzyme secretion is not impaired in the transgenic animals

Fig 3: CFTR knockdown abolishes the effects of CDX2 on resisting hyperglycemia-induced RTECs injury. NRK-52E cells were, respectively, non-transfected (NG group or HG group), and transfected with CDX2-overexpressing / Vector1 (HG+OE-CDX2 group / HG+Vector1 group) or CFTR-knockdown / Vector2 plasmid (HG+Sh-CFTR group / HG+Vector2 group), or co-transfected CDX2-overexpressing + CFTR-knockdown plasmid (HG+OE-CDX2+Sh-CFTR group). (A–C) Immunoblot bands of CDX2 and CFTR, and quantitative data (B, C). (D–I) Immunoblot bands of activated ß-catenin, Snail, E-cadherin, Vimentin, and Col-III; quantitative data are shown (E–I). Data are mean±SD from three experiments performed independently. n=3; *P<0.05 versus HG+Vector1 group; #P<0.05 versus HG+Vector2 group; &P<0.05 versus HG+OE-CDX2 group.

Fig 4: CFTR is a downstream target gene of CDX2, and downregulated in DKD. (A) CFTR promoter activity increased in CDX2-overexpressing NRK-52E cells. NRK-52E cells were administered non-transfected (Con)/pGL3-rno-CFTR-promoter plasmid (pGL3-rno-CFTR), or co-transfected with CDX2-overexpressing/Vector plasmid and pGL3-rno-CFTR-promoter plasmid. n=3; *P<0.05 versus Con group; #P<0.05 versus Vector + pGL3-rno-CFTR group. (B) Immunofluorescent staining of CDX2 and CFTR in NRK-52E cells of the NG and HG groups, respectively (scale bar, 20µm). C-H Western blot bands (C, D) and protein quantitation (E, F) of CFTR in non-transfected (NG or HG treated) NRK-52E cells, and NRK-52E cells transfected with Vector (NG+Vector, HG+Vector), CDX2-overexpressing (NG+OE-CDX2, HG+OE-CDX2) or CDX2-knockdown (NG+Sh-CDX2, HG+Sh-CDX2) plasmid, and mRNA levels (G, H). Data are mean±SD from three experiments performed independently. n=3; *P<0.05 versus NG group; #P<0.05 versus NG+Vector group; &P<0.05 versus HG+Vector group. (I, J) Immunohistochemical staining of CFTR in T1D model mice and controls (J), T2D mice and controls (J). n=6; *P<0.05 versus NC group or db/m group. (K, L) Western blot bands of CDX2 in T1D model mice and controls (K), and quantitative data (L). (M, N) Western blot bands of CFTR in T2D model mice and controls (M), and quantitative data (N). Data are mean±SD from three independent assays. n=6; *P<0.05 versus NC group or db/m group.

Fig 5: Analysis of CFTR and oxidative stress related genes expression (a) Schematic representation of a qPCR device and a plate used for the analysis. (b) ALI-cultured 16HBE14o- cells were exposed to spark-ablated CNP generated with 8 L/min nitrogen gas flow, 1.0 kV gap voltage and 5 mA at day 5 of ALI and gene expression of CFTR, ANO1 and TNFAIP3 was measured by qPCR, normalized to PGK1 expression. Data are mean ± SEM of three independent experiments. Each experiment was measured with two technical replicates. Two-tailed, unpaired student ‘s t-test, * p < 0.05, ** p < 0.01. (c) From the same cells proteins were isolated and 30 µg protein were subjected to western blot analysis (cropped image, depicted are lane 4 and 5 of Fig. S9 with the full western blot image with 3 biological replicates) for CFTR and ß-Actin as reference protein. Data are expressed as fold of CAC samples. Data are mean ± SEM of four independent experiments. Two-tailed, unpaired student ‘s t-test, **p < 0.01. (d) A panel of markers for oxidative stress was analyzed by qPCR. Data are mean ± SEM of three independent experiments. Each experiment was measured with two technical replicates. Two-tailed, unpaired student ‘s t-test, * p < 0.05.