Figure 6

Pkhd1 ^{del3-4/del3-4} collecting duct cells have abnormal endocytosis and membrane protein partition defects. (a) Endocytosis assays in primary confluent cultures of collecting duct cells isolated from control (WT) and Pkhd1 ^{del3-4/del3-4} mouse kidneys (M) using fluorescently-labeled markers for the caveolar [albumin (Alb, red)] and clathrin [transferrin (Tfn, red)] endocytic pathways. Nuclei were stained with DAPI. Scale bars, 50 μm. (b) Quantitation of results for internalization assays using fluorescent markers for the clathrin (Tfn, EGF) and caveolar (alb, LacCer) endocytic pathways in confluent primary cultures of collecting duct cells from control (WT) and Pkhd1 ^{del3-4/del3-4} (M) mice. Data were obtained using conventional fluorescence and are expressed as percent of uptake. Data represent quantification of at least 100 cells from each of three independent experiments. ** P < 0.01, *** P < 0.001. (c) Primary confluent cultures of collecting duct cells isolated from control (WT) and Pkhd1 ^{del3-4/del3-4} mouse kidneys (M) were stained for markers of lysosomes (Lamp2, green) and caveolin-1 containing lipid-raft rich vesicles [Cav1 (red)] and DAPI (blue). The merged image shows co-localization of caveolin-1 with Lamp2 in large, vacuole-like lysosomes in the mutant cells. Scale bars, 10 μm. (d) Liver specimens of 12-week old Crj:CD/SD (Wt) (control) and PCK rats were stained for phospho-Smad3 (p-Smad 3, red), cytokeratin 19 (Ck19, red) and DAPI (blue). Scale bars, 20 μm.(e) Cell lysates of Crj:CD/SD (Wt) (control) and PCK mutant cholangiocyte cell lines treated with TGF-β for the indicated times and fractionated into cytoplasmic (top two panels) and nuclear (bottom two panels) fractions. Immunoblots were probed for phospho-Smad3 (p-Smad3) and total Smad3. Total Smad3 and LaminB1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (f) Tgf-β receptor degradation assay in rat cholangiocyte cell lines. On the left, autoradiographs of a representative experiment for Crj:CD/SD (Wt) (control) and PCK mutant cholangiocyte cell lines treated with [125I]-labeled human TGF-β and then analyzed for levels of labeled Tgf-β receptor subunits at the times indicated. Three separate experiments were carried out, quantified by phosphorimaging and graphed as receptor quantity (% of time 0) vs. time (graph on right). Each point represents the mean ± SD. The results for wild type are in blue and mutant in red. ** P < 0.01. The full-length blots of cropped images in e and f are included in Supplementary Fig. S10.