Figure 2. CpG-TLR9 signaling-induced GSCs requires Frizzled 4/JAK2 interaction.

(A) JAK2 is activated in primary human GSC008 cells upon stimulation by various classes of CpG-ODNs (classes A, B, C; D19 is class A). FACS showing pJAK2 levels in the GSCs receiving either indicated CpG-ODNs or IFN-γ. Ectopic IFN-γ stimulation of GSCs was included as a positive control for inducing JAK2 activation. (B) FACS analysis showing silencing efficacy by Frizzled 4 siRNA introduced into human GSC-like cells. (C) FACS analysis showing expression of activated JAK2 (left panel) or activated STAT3 (right panel) in U87 human glioma cells grown as spheres and transiently transfected with Frizzled4 siRNA, stimulated with CpG-ODN as indicated. (D) Effects of silencing Frizzled 4 (FZD4) on GSC-like cells as phenocopied by sphere formation. U87 human glioma cells were first transfected with indicated siRNAs followed by inducing sphere growth and treatment with CpG-ODN. SD and significance shown: *P ≤ 0.05; **P ≤ 0.01. (E) Phospho-JAK2 and Frizzled 4 co-localize upon CpG-ODN stimulation in GSC-like cells. Upper panels: Shown are representative microscopic images of activated JAK2 and Frizzled 4 co-localization in U87 human glioma cells grown as spheres. The glioma cells were first transiently transfected with either control or Frizzled 4 siRNA, followed by CpG-ODN stimulation. Spheres were analyzed by confocal microscopy. Scale bar 100 μm. Lower panels: Shown are magnified areas in intensity-coded wrong color mode of subcellular localization of Frizzled 4 and pJAK2. Scale bar 10 μm. (F) Frizzled 4 and JAK2 interact in GSC-like cells induced by CpG-ODN. Co-immunoprecipitation analysis shows protein-protein interaction of Frizzled 4 and JAK2 in U87 human GSC-like cells. The tumor cells were transfected with either scrambled siRNA or Frizzled4 siRNA, followed by CpG-ODN stimulation.