Fig. 3.

Chi3l3 induces a pro-oligodendrogenic transcriptional and signaling profile. a, b Gene expression of the pro-oligodendroglial transcription factors Olig1, Olig2, and Sox10 after 36 h a and negative transcriptional regulators Id2, Id4, and Hes5 after 24 h b in differentiating neural stem cells (NSCs) post exposure to Chi3l3 or PBS (control). c Mean fluorescence intensity (MFI) quantification of individual NSCs, activated by Chi3l3 or PBS (control), fixed at 5, 15, and 30 min and immunostaining for phosphorylation (p)- specific antibodies to various signaling proteins. d Ingenuity pathway analysis showing interconnection between Chi3l3-mediated signaling events (Ptk2b (Pyk2), MAPK14 (p38MAPK), PLCG2 (PLCγ2), RAF1 (c-Raf), ERK1/2, PI3K p85 and transcriptional events (Olig1, Olig2, Sox10, Hes5, Id2, Id4). e, f Gene expression of Olig2 in differentiating NSCs in the presence of Pyk2 kinase inhibitor PF-431396 e or NSCs, transfected with control shLVPs or Ptk2b (Pyk2) shLVPs f after 36 h of exposure to Chi3l3 or PBS. g, h Gene expression of Olig2 in differentiating NSCs in presence of MEK-Erk inhibitor U0126 g or NSCs, transfected with LVPs containing GFP (control) or dnErk2-GFP h after 36 h of exposure to Chi3l3 or PBS. Values were normalized to housekeeping genes and expressed as percent change to PBS-treated control NSCs. i Quantification of NG2⁺ oligodendrocyte precursor cells, derived from NSCs differentiated for 3 days in the presence ( + ) or absence (−) of PF-431396 and Chi3l3 ( + ) or PBS (−). j Quantification of NG2⁺ oligodendrocyte precursor cells derived from differentiating NSCs, transfected with control shLVPs or Ptk2b shLVPs (Pyk2), and treated with Chi3l3 ( + ) or PBS (−). Pyk2 signaling deficiency significantly reduced Chi3l3-induced oligodendrogenesis. Values are expressed as percent change to the corresponding PBS-treated group. Two-tailed Student’s t test was performed for a–c, one-way ANOVA followed by Turkey’s post hoc test was performed for e–j; Data are representative of three independent experiments. For n numbers please refer the Methods section; mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.005