Fig. 4

 Cse1l Negatively Regulates Fluid Secretion in Mammalian MDCK-C7 Cells

(Aa) Zebrafish GFP-Cse1l, but not GFP-Cse1l^s866, coimmunoprecipitates with human CFTR-CTHA.

(Ab) Partial colocalization (arrows) of GFP-Cse1l and CFTR-CTHA in transfected HEK293 cells. N indicates nucleus. Scale bar represents 10 μm.

(B) Overexpression of zebrafish GFP-Cse1l abrogates the stimulatory effect of forskolin on CFTR-dependent fluid secretion in 3D cultures of mammalian MDCK-C7 cells.

(Ba) MDCK-C7 cells were grown on Matrigel for 4 days, and forskolin (1 μM) was then added for 16 hr before the cysts were fixed and imaged. Green is GFP, red is F-actin, and blue is β-catenin. Scale bars represent 50 μm.

(Bb) Quantification of data from (Ba) (n = 161 for GFP-Cse1l^s866; n = 131 for GFP-Cse1l). Error bars indicate standard error of the mean.

(C) Depletion of Cse1l leads to increased fluid secretion in 3D cultures of MDCK-C7 cells.

(Ca) Immunoblot showing knockdown of dog Cse1l in cells expressing a shRNA targeting Cse1l (Sh-4) but not in control cells expressing a nonsilencing shRNA (Sh-scr).

(Cb) Knockdown of dog Cse1l leads to lumen expansion in control but not in GFP-Cse1l-expressing MDCK-C7 cells in 3D cultures (5 days in Matrigel) following forskolin treatment. Red is F-actin, green is β-catenin in upper panels and GFP in lower panels, and blue is ToPro. Scale bars represent 50 μm.

(Cc) Quantification of data from (Cb) (n = 200 for control; n = 149 for GFP-Cse1l). Error bars indicate standard error of the mean.