Fig 2: PKD2 has a protective effect on epithelial barrier function in vivo or vitro.(a) PKD2 knockin mutant in vivo decreased the expression of ZO-1and MUC2 in colonic mucosa of PKD2SSAA/SSAA and PKD2WT/WT mice. (b) Plasma fluorescene was measured following oral gavage of mice with FITC-dextran (4 kDa) exposed to DSS. ***p < 0.001 as measured by one way-ANOVA among four groups. (c) Representative images (50×) of immunofluorescence staining performed on frozen sections of colon from DSS-treated mice exposed to orally administered FITC-dextran (4 kDa) shows FITC permeation into lumen (green) is enhanced in PKD2SSAA/SSAA mice treated with DSS compared with wild-type controls. Nuclei are stained with DAPI (blue). Bar represents 2 µm. (d) PKD specific inhibitors increases permeability of Caco-2 cells to FITC-dextran. Results represent three independent experiments with two mice per group.

Fig 3: The expression of osteoblastic differentiation markers were inhibited by PKD1 deficiency.(a,b) IHC staining of PKD1 and osteoblastic markers OCN and OPN in femur from 4-week KO and control mouse. Positive osteoblasts were showed by black arrows. (c) Number of PKD1 positive, osteopontin positive (N.OPN+) and osteocalcin positive (N.OCN+) osteoblasts on the bone surface were measured as positive cells per millimeter of perimeter in sections (B.Pm). Measurements were presented as mean ± SD. *p < 0.05 versus respective control. The data in each group were analyzed using unpaired, two-tailed Student’s t-test. (d) Relative mRNA expression levels of osteoblast differentiation markers (ALP, OSX, Runx2, and col-α1) and osteoclast marker (TRAP) in long bone from wild-type and PKD1-KO female mice. The level of each marker gene expression was normalized to the level of the housekeeping gene. n > 3, WT and KO. *p < 0.05.

Fig 4: CRT0066101 inhibits PKD activity in osteoclasts. (A) Preosteoclasts were pre-treated with increasing doses of CRT0066101 for 30 minutes prior to stimulation with 50 mM phorbol 12-myristate 13-acetate (PMA), indicated by ‘+’ for 5 minutes. (B) Quantitation of P-Ser916 levels relative to the level from PMA-alone set as 1.0. * p < 0.05 versus PMA alone. (C) Treatment of preosteoclasts with the indicated CRT0066101 doses for 30 minutes and blotted against endogenous P-Ser916. (D) Quantitation of experiments from (C) with the level from untreated cells set as 1.0. * p < 0.05 versus control. Each experiment was performed four times. Representative Western blots from single experiments are shown, while the graphs represent the collective results from the independent experiments analyzed together as described in Section 4.12.

Fig 5: Immunofluorescence localization of PKD2 and PKD3 in pre-osteoclasts and early post-fusion osteoclasts. Osteoclast cultures at preosteoclast and early multinucleated osteoclast stages of differentiation were stained with the indicated PKD antibodies (green), rhodamine-phalloidin (for F-actin, red) and DAPI (for DNA, blue) and imaged by confocal microscopy. Merged images are shown above and the corresponding green channel below. Scale bar = 20 µm. Each of these antibody stainings and timepoints has been performed three independent times with similar results.