NIK mRNA is up-regulated by TWEAK and NIK protein overexpression promotes tumorigenesis. (A) Quantification of BT114 invasion density at 48 h with or without 10 ng/ml TWEAK in the collagen matrix. Data represent average numbers of invading cells per 1-mm2 field (n = 3 wells) ± S.E.M. ***p < 0.001 relative to vehicle control using unpaired Student’s t-test. (B) qRT-PCR analysis of NIK mRNA expression from BT114 cells. mRNA was collected at 48 h after stimulation with 10 ng/ml TWEAK or vehicle control. Data represent average NIK mRNA expression relative to RPLP0 mRNA and normalized to untreated control (n = 3 replicates) ± S.D. ***p < 0.001 relative to vehicle control using unpaired Student’s t-test. (C) Western blot analysis of BT114 glioma cells transduced with lentivirus containing NIK cDNA or luciferase cDNA (Con) using the indicated antibodies. (D) Representative IVIS images of 3-D rendering of tumors derived from DiD-stained intracranial-injected BT114-control (n = 7 mice) or BT114-NIK (n = 9 mice) cells at 14 and 24 days post-injection (n = 3 mice per group). Numbers indicate scale of Signal Intensity for each image. (E) Quantification of BT114 brain tumors in Figure 5D at 30 days. Data represent average tumor volume ± S.E.M. *p < 0.05 relative to BT114 control using unpaired Student’s t-test. (F) Representative IVIS ex-vivo images of DiD-stained BT114 brain tumors in Figure 5D at 58 days post-injection.