Figure 3
HGF treatment promotes apical localization of VX-809-rescued Phe508del-CFTR in polarized CFBE cells. (a) WB analysis of whole cell lysates from polarized CFBE cells treated for 48 h with either DMSO or 3 μM of VX-809 in the presence or absence of HGF for the last 24 h, as indicated, and stimulated acutely with 10 μM of VX-770 (acVX-770). Shown are representative images of CFTR and Ki-67 immunoblots together with bar plots of band intensity quantification, normalized to DMSO (means ± SEM), from three independent assays. Tubulin was used as a loading normalizer in band intensity quantification. (b) Immunofluorescence staining of polarized CFBE-Phe508del cells treated as in (a), were stained with anti-CFTR/Alexa 488, phalloidin-TRITC and DAPI, and analysed by confocal microscopy. Shown are overlay images (upper images) as well as isolated CFTR-staining (green channel) representative of the indicated treatment conditions. Yellow trace lines exemplify the method used for CFTR signal quantification. Actin signal was used as a guide to define the apical (AP) and basolateral (BL) membrane regions (dotted lines) that were used to quantify CFTR signal intensity (solid lines). AP, BL and Total (BL + AP) signal intensities in cells treated or not with HGF and VX-809/acVX-770 are plotted in (c) and the ratio between AP and BL signal intensities in (d). Data indicates mean ± SEM of signals from at least 30 cells analysed in each of the three independent experiments. White arrows indicate apical shift in CFTR localization upon HGF treatment. White horizontal bar represents 10 μm. #p < 0.001 (e) Conventional cell surface protein biotinylation assays of CFBE cells treated as in (a). Fold change in total (WCL) and PM (Surface)-rescued Phe508del-CFTR band C levels were quantified by WB densitometry and are indicated below the respective panels.
