Figure 1

Fibrocystin/Polyductin (FPC) regulates RhoA levels and function. (a) Immunoblots of total cell lysates of multiple independently-derived PKHD1-expressing and pcDNA5 vector control (cont) Flp-In MDCK cell lines probed for RhoA, active RhoA, Rac1 and Cdc42. The blots were stripped and re-probed for actin as a loading control (except the active RhoA blot). (b) On the left are immunoblots of cell lysates from IMCD cell lines stably expressing siRNA targeting either Pkhd1 (kd) or a random sequence (cont). The bar graphs on the right provide the quantitative analysis of three independent experiments. The level of expression of each Rho GTPase was determined relative to that of actin or tubulin, and the values for the control cell line was arbitrarily set at one. For the active RhoA blot, equal amounts of cell lysate were loaded. Values represent mean ± s.d. **P < 0.01. (c) Same as in panel “b” except primary cultures of collecting duct cells isolated from wild type controls (WT) or Pkhd1 ^{del3-4/del3-4} (M) were the source material. Values represent the mean ± s.d. of three experiments, **P < 0.01. (d) RhoA was immunoprecipitated from cell lysates of primary cultures of wild type (WT) and Pkhd1 ^{del3-4/del3-4} (M) collecting duct cells and control (C) and Pkhd1-silenced IMCD (kd) cells and then immunoblotted for ubiquitin (top panel) and RhoA (middle panel). Mouse IgG was used as a negative control (IgG). Equal amounts of cell lysate input were immunoblotted for actin (bottom panel).(e) Kidney of control (WT) and littermate Pkhd1 ^{del3-4/del3-4} (Mutant) mice stained with phalloidin (red) and DAPI (blue). Scale bars, 20 μm. (f) Kidney specimens from human control (control) and two different ARPKD donors (ARPKD1, ARPKD2) stained for Dolichos Biflorus Agglutinin (DBA), a marker for collecting ducts (grey), phalloidin (red) and DAPI (blue). Scale bars, 10μm. (g) Bile ducts of control (WT BD) and littermate Pkhd1 ^{del3-4/del3-4} (Mutant BD) mice stained with phalloidin (green) and DAPI (blue). Scale bars, 20 μm. (h) Cholangiocyte cell line derived from PCK rat treated with either scrambled control or RhoA siRNA and stained with DAPI (blue) and phalloidin (red). Scale bars, 20 μm. (i)Scatter plot of the fluorescent intensity for studies represented in “h”. Asterisk denotes p < 0.05. (j)Representative western blot of lysate from cell cultures described in “h” and probed for RhoA and beta-actin as a loading control. Active RhoA levels are displayed in bottom panel. The full-length blots of cropped images in a-d and j are included in Supplementary Figure S7.