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. 2008 Jan 22;28(7):2368–2379. doi: 10.1128/MCB.01780-07

FIG. 6.

FIG. 6.

Dkk1-induced neurite outgrowth and LRP5/LRP6 siRNA-induced neurite outgrowth were blocked by sFRP-1. (A) sFRP-1 blocked Dkk1-induced neurite outgrowth. TC-32 cells plated on coverslips were pretreated with sFRP-1 (10 μg/ml) for 30 min at 37°C in a CO2 incubator and subsequently incubated in the presence or absence of Dkk1 (1 μg/ml) for 3 h. Quantitative analysis of neurite outgrowth was performed as described for Fig. 5C. The results are means ± standard deviations (error bars) from three independent experiments. **, P < 0.01. (B) sFRP-1 blocked LRP5/LRP6 siRNA-induced neurite outgrowth. TC-32 cells were transfected with LRP5/LRP6 or Luc control siRNA and were plated on coverslips. Approximately 48 h after transfection, medium was replaced with serum-free RPMI with or without sFRP-1 (10 μg/ml), and cells were then incubated for 6 h at 37°C in a CO2 incubator. Quantitative analysis of neurite outgrowth was performed as described for Fig. 5F. The results are means ± standard deviations (error bars) from three independent experiments. *, P < 0.05. (C) The effect of sFRP-1 on LiCl-stimulated neurite outgrowth. TC-32 cells plated on coverslips were pretreated with sFRP-1 (10 μg/ml) for 30 min at 37°C in a CO2 incubator and further incubated with LiCl (20 mM) or negative control NaCl (20 mM) in serum-free RPMI for 3 h. The percentage of cells having neurites was determined for each treatment group. The stimulation of neurite outgrowth for each group was defined as the ratio of this percentage to the percentage observed for the NaCl control. The results are means ± standard deviations (error bars) from three independent experiments. ***, P < 0.001. (D) Fzd3 siRNA blocked Dkk1-dependent neurite extension. TC-32 cells were transfected with Fzd3 siRNA and plated on coverslips. Approximately 48 h after transfection, medium was replaced with serum-free RPMI with or without Dkk1 (1 μg/ml) and cells were incubated for an additional 3 h. The results are means ± standard deviations (error bars) from three independent experiments. −, absence of; +, presence of; **, P < 0.01.