VGF protein is associated with TMs, without an obvious impact on function. a, Western blot analysis of VGF and Ttyh1 protein expression levels of S24 and T1 GBMSCs cultured under serum-free, nonadherent conditions and serum-containing, adherent conditions for 7 d, demonstrating a downregulation on the protein level under adherent conditions (cropped gels). b, Maximum intensity projection images of immunohistochemical stainings of human nestin (green) and VGF (red) acquired by confocal microscopy (S24 GBMSC tumors). Arrowheads indicate VGF-positive TMs. c, Verification of shRNA knockdown of VGF in S24 GBMSC by Western blot analysis (95% reduction in protein level). d, In vivo 3D MPLSM images of S24 shControl and shVGF GBMSCs on day 40 (z = 45 μm), demonstrating no obvious phenotypical aberration and no apparent difference in tumor invasion and growth. Green, GBMSCs; blue, blood vessels.