Skip to main content
. 2017 Jul 12;7:5233. doi: 10.1038/s41598-017-05435-5

Figure 4.

Figure 4

Suppression of CFTR activates β-catenin signaling pathway by interacting with DVL-2 in renal epithelial cells. (a) Western blot showing the increased nuclear accumulation of β-catenin in ribozymes-mediated CFTR knockdown HK-2 cells (Rib) comparing to control cells (His) transfected with control vectors (pEF6/V5-His); (N.E-nuclear extraction; C.E-cytoplasmic extraction. (Full-length blot is shown in Supplementary Figure S7g.) (b) luciferase activity assay showing knockdown of CFTR increases β-catenin transcriptional activity whereas overexpression of CFTR (pcDNA3.1 CFTRov) decreases β-catenin activity in HK-2 cells. Cells were lysed at 48 hours after transfected with TOPflash reporter gene (0.5 μg/ml) and Renilla-Luc (0.05 μg/ml) and the relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for renilla luciferase with that of control group set as 1.0. *p < 0.05; (c) Real time-PCR showing increased mRNA expression of β-catenin target genes in CFTR knockdown HK-2 cells, **p < 0.01; (d) Western analysis showing increased protein expression of β-catenin target genes in the CFTR knockdown HK-2 cells. CFTR knockdown HK-2 cells (Rib) comparing to control cells (His) transfected with control vectors (pEF6/V5-His); (Full-length blot is shown in Supplementary Figure S7h.) (e) Western blot analysis shows that iCRT14 reverses decreased expression of E-cadherin in a dose-dependent manner in CFTR knockdown HK-2 cells; (Full-length blot is shown in Supplementary Figure S7i.) (f) Immunofluorescent staining showing the co-localization of CFTR and Dvl2 in HK-2 cells; (g) Co-immunoprecipitation showing the interaction of CFTR and Dvl2 in HK2 cells; (Full-length blot is shown in Supplementary Figure S7j.) (h) CO-IP of exogenous CFTR and Dvl2 in HEK293 cells showing lack of interaction when CFTR PDZ-binding domain is deleted; (TL: total cell lysates; full-length blot is shown in Supplementary Figure S7k.) (i) luciferase activity assay shows that overexpression of CFTR wild type (peGFP-CFTR), but not CFTR with deleted PDZ-binding domain (peGFP-CFTR delPDZ), increases β-catenin transcriptional activity in HK-2 cells. Cells were lysed at 48 hours after transfected with TOPflash reporter gene (0.5 μg/ml) and Renilla-Luc (0.05 μg/ml) and the relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for renilla luciferase with that of control group set as 1.0. *p < 0.05.