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. 2010 Sep 13;5(9):e12682. doi: 10.1371/journal.pone.0012682

Figure 2. LRP6 antibodies can have different effects on autocrine Wnt signaling in other cell lines.

Figure 2

(A) Summary table of 11 cell lines displaying autocrine Wnt signaling inhibited by Fzd8CRD-Fc protein (10 µg/ml), and the effects of LRP6 antibodies on autocrine signaling. The fold change in autocrine Wnt signaling is highlighted in red or green to indicate inhibition or potentiation, respectively, with p-value <0.01 by one-way analysis of variance (ANOVA). Antibodies were used at 10 µg/ml, except for NCI-H358 and HT-1080 cells which were treated with 1 µg/ml YW211.31.57 or YW210.09 antibody, respectively, to increase potentiation effects. (B) qPCR expression analysis of AXIN2 mRNA in four cell lines treated with 25 µg/ml YW211.31.57 antibody or Fzd8CRD-Fc protein, with and without (NA) 0.2 µg/ml Wnt3a. (C–D) Expression of Wnt-induced genes in NCI-H23 (C) and M14 (D) cells is potentiated by YW211.31.57 and antagonized by YW210.09 antibody (30 µg/ml). CD4-Fc protein (30 µg/ml) serves as a negative controls (C). For M14 cells (D), AXIN2 and SP5 expression is potentiated more potently by Wnt3a protein or YW211.31.57 antibody than is APCDD1 and ZNRF3 expression. (E–F) In Hs578T cells stably integrated with Wnt luciferase reporter, YW211.31.57 antibody shows concentration-dependent inhibition of Wnt3a-stimulated signaling (E) and potentiation of autocrine Wnt signaling (F). Fzd8CRD-Fc protein inhibits and YW210.09 antibody potentiates signaling with or without 0.1 µg/ml Wnt3a stimulation. RNAi experiments indicate that at least 41% of Wnt3a-induced signaling in Hs578T cells is dependent on LPR5 expression (data not shown), and this signaling is predicted to be inhibited by Fzd8CRD-Fc protein but not YW211.31.57 antibody. In this experiment, SV40-driven luciferase was not transfected for normalization and, instead, antibody and protein treatments were independently confirmed to have no significant effect on viability of this cell line. (G–H) EKVX cells transfected with Wnt luciferase reporter also display potentiation of autocrine Wnt signaling (NA) and antagonism of Wnt3a-induced signaling by YW211.31.57 antibody. Antibody-mediated potentiation of autocrine Wnt signaling is inhibited by 5 µg/ml Fzd8CRD-Fc protein (H).