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. 2024 Nov 15;27(12):111393. doi: 10.1016/j.isci.2024.111393

Figure 6.

Figure 6

CFTR protein presence, but not function, regulates PDX1 expression

(A) Short circuit current measurement in WT PDE cultures differentiated in the presence of CFTR inhibitor GlyH101 or vehicle (DMSO). Responses to sequential addition of amiloride, DIDS, IBMX/Forskolin (IF), and GlyH101 are shown (n = 4 donors with 2 cultures averaged per donor). Inset is a representative current trace for each condition.

(B) RT-qPCR quantification of PDX1 expression under the conditions show in (A). PDX1 expression in CFTR-KO PDE cultures without GlyH101 is shown for comparison (n = 4 donors with 3–4 cultures combined for RNA).

(C) PDX1 expression in PDE cultures derived from ferret PDCs with homozygous CFTR-G551D germ line and differentiated in the presence of VX-770 (+) or DMSO (−) (n = 3 donors with 3–4 cultures combined for RNA). PDX1 expression in CFTR-KO PDEs is shown for comparison (n = 3 donors with 3–4 transwells combined for RNA). PDX1 expression is normalized to that of (B) WT (−) or (C) G551D (−).

(D) Schematic of human CFTR (hCFTR) complementation in CFTR-KO PDCs using a lentiviral vector that also expresses tdTomato. A vector expressing just tdTomato was used as a control (cont). FACS-enriched tdTomato-positive cells were used to generate PDE cultures. Representative images of PDC cultures before and after FACS enrichment are shown below the schematic.

(E) Short circuit current measurements of PDE cultures-derived lentiviral transduced CFTR-KO PDCs expressing hCFTR/tdTomato or tdTomato alone. Responses to sequential addition of amiloride, DIDS, IBMX/Forskolin (IF), and GlyH101 are shown (n = 3 donors). Inset is a representative trace of current for each condition.

(F) RT-qPCR quantification of ductal (SOX9, HNF6) and endocrine (PDX1, PAX6, NKX6.1) genes expression from the conditions in (E) (n = 3 donors with 2 cultures combined for RNA). Expression levels are normalized to CFTR-KO tdTomato PDE cultures.

(G) Immunofluorescence images of PDX1 and INS expression in 2-month-old WT ferrets and CFTR mutants ferrets with variable expression of CFTR protein (percent CFTR expression is shown on top of each image). Images were obtained on confocal microscope Zeiss 880 at 20X magnification and processed for maximum intensity projection. Scale bars, 50 μm.

(H) Quantification of PDX1 expression in ductal epithelium (n = 3 donors for each genotype). All graphs show the mean ± SEM. Significance was calculated using nonparametric Mann-Whitney t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).