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. 2013 Jan 9;110(4):1440–1445. doi: 10.1073/pnas.1211179110

Fig. 4.

Fig. 4.

DKK-1 inhibits PDGF-BB–mediated proliferation of pericytes in vitro by a noncanonical, LRP-6–dependent, P42/44 MAPK-dependent mechanism. (A) Graph of BrdU incorporation into quiescent kidney pericytes 6 h after stimulation with cytokines. (B) The effect of DKK-1 on PDGF-BB–stimulated proliferation. (C) qPCR of genes associated with cell activation in pericytes 48 h after stimulation. (D) Quantification of cell viability in pericytes stimulated with cytokines and DKK-1 for 24 h. (E) RT-PCR results showing the effect of PDGF-BB on WNT ligands and receptors in pericytes 12 h after stimulation. C, control; P, PDGF; arrowheads indicate regulated genes. (F) Western blot time course showing pPDGFRβ and pLRP-6 levels in pericytes. DKK-1 does not affect pLRP-6 at early time points but inhibits pLRP-6 at later time points. (G) Fluorescence images and data quantifying nuclear GFP+ (green) in TCF/Lef:H2B-GFPTr canonical WNT reporter pericytes 16 h after PDGF-BB or PDGF-BB + DKK-1. (H) Western blot time course of phosphorylated forms of P42/P44, JNK, and P38, PDGFRβ, and total cyclinD1 in pericytes activated by PDGF-BB or PDGF-BB + DKK-1. (I) Graph showing the effect of DKK-1 or the canonical WNT inhibitor XAV939, the P42/P44 inhibitor U0126, or the JNK inhibitor SP600125 on PDGF-BB–stimulated BrdU incorporation into quiescent pericytes. (J) Graph showing the effect of PDGF-BB on the proliferation of Ctnnb1fl/fl pericytes that underwent in vitro recombination by expressing Cre recombinase vs. Ctnnb1fl/fl pericytes that expressed control protein GFP. (K) Western blot of pericyte proteins immunoprecipitated by anti-PDGFRβ antibodies or control antibodies detecting pLRP-6 or PDGFRβ. (L) Graph showing the effect of expression of LRP-6 (wild type) or dominant-negative forms of LRP-6, LRP-6 with tyrosine-to-methionine mutations at the five tyrosine sites (5m), or LRP-6 lacking the cytoplasmic tail (ΔC) on 3T3 fibroblast proliferation in response to PDGF-BB and DKK-1. *P < 0.05, **P < 0.01, ***P < 0.01. n = 4–7 per group. All blots are representative of three experiments. (Scale bars, 25 µm.) Error bars indicate SEM.