Figure 4. Wnt5a regulate VEGF-A₁₆₅b expression via Ror2/JNK/SC35-dependent mechanism in macrophages.

a. VEGF-A₁₆₅b protein expression was measured by western immunoblot analysis in the presence or absence of recombinant Wnt5a in cultured RAW264.7 cells after treatment for 24 hours. b. Vegfa₁₆₅b mRNA expression was measured by qRT-PCR 8 hours after treatment with Wnt5a (500 μg/ml) or vehicle in RAW264.7 cells that had been pre-incubated with vehicle, sh-control or sh-Ror2. ANOVA with post-hoc Tukey HSD. c. Phosphorylation of JNK (p-JNK) was measured by western blot analysis in cells treated with or without recombinant Wnt5a for 30 minutes. Relative phosphorylation levels of JNK were quantified by using ImageJ. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. d. The effects of JNK inhibition and Sfrp5 treatment or Wnt5a-induced Vegfa₁₆₅b expression. Vegfa₁₆₅b mRNA expression was measured by qRT-PCR. SP600125 was added 10 minutes prior to the addition of recombinant Wnt5a (500 ng/ml). Recombinant Sfrp5 was added at 1 μg/ml. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. e. SC35 protein expression was measured by western blot analysis in the presence or absence of stimulation with recombinant Wnt5a. The effects of the JNK inhibitor SP600125 or Sfrp5 on Wnt5a-induced SC35 expression were evaluated at 1 hour. Relative levels of SC35 were quantified by using ImageJ. Immunoblots were normalized to LaminA/C signal. Results are shown as the mean ± S.E. (n = 3/group). ANOVA with post-hoc Tukey HSD. f. SC35 is essential for Vegfa₁₆₅b expression. Vegfa₁₆₅b mRNA expression was measured by qRT-PCR. Cells were treated with sh-control and sh-SC35 for 8 hours and treated with or without recombinant Wnt5a. ANOVA with post-hoc Tukey HSD. g. Graphic scheme of proposed mechanism. *P < 0.05, **P < 0.01, ***P < 0.001.