Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Endogenous Chi3l3 expression in the adult SVZ during EAE. a Chi3l3 gene expression in the subventricular zone (SVZ) of healthy control mice (HC; n = 8), preclinical EAE (6 days post immunization (dpi; n = 6), EAE onset (13dpi; n = 6), acute EAE (14 dpi; n = 6), initial recovery (18 dpi; n = 8) and chronic EAE (36 dpi, n = 6) measured by qRT-PCR (multiple t -tests with Dunn–Bonferroni’s multiple comparison test). b – e Confocal images of the SVZ of representative healthy controls (HC), acute EAE mice (12–14 dpi). Sections were immunostained for the nuclear marker TO-PRO-3 (blue), Chi3l3 (red) and the microglia/macrophage marker CD11b (green, b – e ) and the lymphocyte common antigen CD45 (blue, d ). Chi3l3 expression in the SVZ was only detected during acute EAE and was localized to CD11b-positive cells (yellow arrowheads) within the corpus callosum (CC) c and lesions d , where they co-expressed CD45 at high (yellow arrowhead) and low levels (white arrowhead). Dashed lines mark the wall of the lateral ventricle (LV). Scale bar, 200 μm. e Ortho-view images of representative Chi3l3 + /CD11b + double-positive cells exhibiting round (upper panel) and ramified (lower panel) morphology. (mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Expressing, Quantitative RT-PCR, Marker

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: In Vitro, Cell Culture, Staining, Marker, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: 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

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Expressing, Fluorescence, Immunostaining, Transfection, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 promotes oligodendrogenesis by inducing EGFR signaling. a ingenuity pathway analysis showing membrane-bound receptors (EGFR, FGFR1, DLG4 (PSD95), NTRK2, FYN) upstream of Chi3l3-induced MAPK signaling (Ptk2b (Pyk2), MAPK14 (p38MAPK), PLCG2 (PLCγ2), RAF1 (c-Raf), ERK1/2, PI3K p85) and pro-oligodendrogenic transcriptional pattern ( Olig1, Olig2, Sox10, Hes5 , Id2 , Id4 ). b Mean fluorescence intensity (MFI) quantification of individual neural stem cells (NSCs), activated by Chi3l3, EGF, or PBS (control) for 15 min, and immunostaining for phospho (p)-specific EGFR (Tyr1068). One-way-ANOVA with Dunn’s multiple comparison test; c Quantification of NSC-derived NG2 + oligodendrocyte precursor cells after 3 days of differentiation in the presence of Chi3l3 ( + ) or PBS (−) and EGFR kinase inhibitor AZD8931 ( + , left) and Gefitinib ( + , right) or DMSO (−). One-way ANOVA with Bonferroni’s multiple comparison test; d Quantification of NG2 + oligodendrocyte precursor cells derived from differentiating NSCs, transfected with control shLVPs or EGFR shLVPs and treated with Chi3l3 ( + ) or PBS (−). Deficiency in EGFR signaling significantly reduced Chi3l3-induced oligodendrogenesis. One-way ANOVA with Bonferroni’s multiple comparison test; e Mean fluorescence intensity (MFI) quantification of NSCs, activated by Chi3l3 ( + ), EGF ( + , positive control) or PBS (−, negative control) in the presence of EGFR inhibitor AZD8931 for 15 min, and immunostaining for phospho (p)-specific Pyk2 (Tyr402). One-way ANOVA with Bonferroni’s ( d , neuroblasts, red) test; f Mean fluorescence intensity (MFI) quantification of NSCs transfected with control shLVPs or EGFR shLVPs, activated by Chi3l3 ( + ), EGF ( + , positive control) or PBS (−, negative control for 15 min, and immunostained for phospho (p)-specific Pyk2 (Tyr402). Deficiency in EGFR signaling reduced Pyk2 phosphorylation in neural stem cells. All values are expressed as percent change to PBS-treated NSCs. One-way ANOVA with Bonferroni’s multiple comparison test; mean ± s.e.m; * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Fluorescence, Immunostaining, Derivative Assay, Transfection, Positive Control, Negative Control

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 infusion induces pro-oligodendrogenic fate of subventricular zone neural stem cells in vivo. a Protocol of BrdU labeling via intraperitoneal (i.p.) injection and Chi3l3 administration. b – f Representative confocal images b , d and quantification c , e , f of the ipsilateral (IL) or contralateral (CL) subventricular zone (SVZ) of naive mice infused with Chi3l3 or PBS (vehicle) and immunostained for BrdU (green) and NG2 ( b , oligodendrocyte precursor cells, red) or Dcx ( d , neuroblasts, red). IL SVZ of Chi3l3-treated mice were compared with IL SVZ of vehicle-treated mice as well as the CL site of the same mouse. Dashed lines mark the ventricular wall of the right or left lateral ventricle (RLV; LLV). Scale bar (left), 50 μm. Double-positive cells are indicated by yellow and white arrowheads and representative cells (box) are shown as high magnification images (right). Scale bar (right), 5 μm. Quantifications show a significantly higher percentage of BrdU/NG2 double-positive cells c and reduced percentage of BrdU + /Dcx + cells e in the ipsilateral SVZ of Chi3l3 infused mice indicating pro-oligodendrogenic modulation of SVZ stem cell fate choice in vivo. f Quantification of the total number of BrdU labeled cells/mm 2 in the SVZ showed no significant difference between Chi3l3 or vehicle infusion in the ipsilateral or contralateral SVZ. (One-way ANOVA with Turkey’s post hoc test; c n = 2 mice (Control IL, CL), n = 4 (Chi3l3 IL), and n = 3 (chi3l3 CL); e n = 2 mice per group, n = 4 mice per group; mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: In Vivo, Labeling, Injection

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Subventricular zone-derived Chi3l3 affects EAE severity and oligodendrogenesis. a Protocol of shRNA lentiviral particle (LVP) administration, BrdU injection, and EAE induction. b Clinical score (left) and linear regression curves (right) of mice treated with control shRNA LVPs (black line) or Chi3l3 shRNA LVPs (red line) and immunized with PLP 139–151 . Dashed lines indicate 95% confidence intervals. ( n = 23 control shRNA LVP and 27 Chi3l3 shRNA LVP mice per group; two-way ANOVA; c Quantifications of percentage (left) and number (right) of NG2 + /BrdU + double-positive cells in the ipsilateral (IL) and contralateral (CL) SVZ of Chi3l3 shLVP and control shLVP mice killed at onset of disease in vivo. ( n = 4 per group; two-tailed Student’s t test; d Quantifications of and number of NG2 + cells in the ipsilateral (IL) and contralateral (CL) SVZ of Chi3l3 shLVP and control shLVP mice sacrificed during chronic EAE ( n = 8 control shRNA mice, n = 6 Chi3l3 shRNA mice; two-tailed Student’s t test. e , f Localization (left) and quantification of inflammatory lesions in brain sections (middle) and spinal cord sections (right) of Chi3l3 shLVP and control shLVP mice killed at EAE onset ( e ; n = 4 mice control shRNA, n = 5 mice Chi3l3 shRNA (left, brain), n = 5 mice per group (right, spinal cord; two-tailed Student’s t-test; mean ± s.e.m) and during chronic EAE ( f , n = 7 mice control shRNA mice, n = 8 Chi3l3 shRNA mice (left, brain) and n = 8 mice control shRNA mice, n = 6 Chi3l3 shRNA mice (right, spinal cord); two-tailed Student’s t test). Schematic diagram showing localization of inflammatory lesions of individual mice, each color represents an individual mouse. (mean ± s.e.m.; * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: Derivative Assay, shRNA, Injection, In Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Proposed model of Chi3l3-induced oligodendrogenesis. In the central nervous system, Chi3l3 is expressed and secreted by alternatively activated myeloid cells (MC), including resident microglia and infiltrating macrophages. These cells are located in the corpus callosum (CC) and in close proximity to the subventricular zone (SVZ) of the lateral ventricle (LV). Chi3l3 binds to epidermal growth factor receptor (EGFR) on the surface of endogenous neural stem cells (NSCs) and activates MEK/ERK signaling and a pro-oligodendrogenic transcription factor pattern in a Pyk2-dependent manner, leading to oligodendrogenesis

Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

Techniques: