Fig. 6: EMT-inducing transcription factors (EMT-TFs) are increased in the absence of functional CFTR, with TWIST1 mediating mutant CFTR driven EMT.

A Transcript analysis of EMT-TFs levels by qRT-PCR on different control and F508del homozygous lung samples. Fold-change values were calculated by applying the ΔΔCT method and are represented by mean ± SEM relative to the control samples (n = 3, unless indicated by (2) or (4)). Asterisk indicates significant difference between F508del/F508del and Ctrl samples (unpaired t-test, p < 0.05). B Representative images of native human bronchial tissue (Ctrl and CF) immunostained for Snail + Slug and ZEB1. Nuclei are depicted in blue and the TFs in magenta. Scale bar represents 25 μm. Increased Snail + Slug and ZEB1 levels are present in CF tissues when compared to controls. Apical and basal (A and B respectively) sides of the epithelia are identified and are the same in all pictures. Images are displayed as maximum image projections (MIPs). The tissues stained display secondary/tertiary bronchi and were as similar as possible for comparison. The CF lung in this figure had a R347P/711 + 5 G > A genotype, but similar findings were obtained in lungs with a F508del/F508del genotype. Several controls were also assessed with similar results between themselves. (n = 2–3 samples). C, D Representative western blots showing Snail + Slug and TWIST levels on C fully differentiated pHBE cells and D polarized CFBE cells. Calnexin, tubulin or GAPDH were used as loading controls. E Representative Western blots showing the effects of VX-445, VX-661 and/or VX-770 on TWIST1 and Snail + Slug in polarized CFBE cells. Calnexin was used as a loading control. F Quantification by densitometry of the protein expression detected by WB in C. Data is normalized to loading control and to Ctrl cells and showed as arbitrary units (A.U.), mean ± SEM. The CF individuals’ genotypes were F508del/F508del, R347P/711 + 5 G > A and M1101K/1609delCA. (n = 3). G Quantification by densitometry of the protein expression detected by WB in D. Data is normalized to loading control and showed as arbitrary units (A.U.), mean ± SEM. Asterisk indicates significant difference between wt-CFTR and F508del-CFTR cells (unpaired t-test, p < 0.05). (n = 3). H Quantification by densitometry of the protein expression detected by WB in E. Data is normalized to loading control and to negative control (DMSO) and showed as arbitrary units (A.U.), mean ± SEM. Asterisk indicates significant difference between negative control and treatment (unpaired t-test, p < 0.05). (n = 4). I Representative Western blots showing the effects of TWIST1 knockdown on CFTR, E-cadherin, N-cadherin, CK18 and vimentin in polarized CFBE cells. Calnexin was used as a loading control. J Quantification by densitometry of the protein expression detected by WB in I. Data is normalized to loading control (for E-cad., CK18, N-cad. and Vim) or to loading control and negative control (TWIST1) and showed as arbitrary units (A.U.), mean ± SEM. Asterisk indicates significant difference between shLuciferase and shTWIST1, hash indicates significant difference between NCtrl and TGF-β1 (unpaired t-test, p < 0.05). (n = 3).