Wisp 1

The brain tissue was fixed with 4% paraformaldehyde at 4°C, after the brain tissues sink to the bottom of the bottle and after dehydrating by concentration of gradient (10%, 20%, 30%, and 5%), and then, 12 μm frozen sections were prepared and examined. All sections were blocked with blocking solution (10% goat serum, 3% BSA, and 0.1% Triton X-100) for 1 h at 37°C, incubating overnight with antibody WISP1 (diluted 1 : 500; Santa Cruz), anti-GFAP (1 : 400; BD Pharmingen), anti-NeuN (1 : 1000; Abcam), β-catenin (1 : 500; Santa Cruz), anti-cleaved caspase-3 (1 : 400; Cell Signaling Technology), anti-LC3 (1 : 500; Cell Signaling Technology), and anti-Iba1 (1 : 1000; Wako) and then washing them with 0.01 M PBS for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again in PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope (Leica DM 5000B, Germany).

Recombinant human WISP-1 and VEGF-A were purchased from PeproTech (Rocky Hill, NJ, USA). α-Tubulin, JNK, FAK, HIF-1α, WISP-1, VEGF-A, p-JNK, and p-FAK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SP600125, HIF i, and FAK i were purchased from Calbiochem (San Diego, CA, USA). Smartpool ON-TARGET siRNAs of control, JNK, FAK, and HIF-1α were purchased from Dharmacon Research (Lafayette, CO, USA). The MiRNome microRNA Profilers QuantiMir kit was purchased from System Biosciences (Mountain View, CA, USA). Mimics of control and miR-381 were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-FAK, FAK, p-c-Src, c-Src, p-EGFR, EGFR, p-ERK, ERK, HIF1-α, β-actin, CD31, and WISP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); VEGF-A antibody was purchased from Abcam (Cambridge, MA, USA). Recombinant human VEGF-A was purchased from R&D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium (DMEM), F-12 medium, fetal bovine serum (FBS) and all other cell culture reagents were from Gibco-BRL Life Technologies (Grand Island, NY, USA). ON-TARGETplus siRNAs were purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were from Sigma-Aldrich (St Louis, MO, USA). The efficacy of all inhibitors were provided in supplementary information (Figure S6). The methods of Transwell migration assay, Western blot analysis, Quantitative real-time PCR, Enzyme-linked immunosorbent assay, Tube formation, Establishment of the WISP-1 knockdown SCC4 cell line and Chick chorioallantoic membrane assay are provided in Supplementary Information.