MMPs are required for glioma invasion and TWEAK-induced MMP9 expression is p52-dependent. (A) Quantification of invasion density at 48 h for BT116 cells with or without 10 ng/ml TWEAK in the collagen matrix. Cells were treated during the 48 h invasion assay with broad-spectrum MMP inhibitor GM6001 at indicated concentrations. Data represent average numbers of invading cells per 1-mm2 field (n = 3 wells) ± S.E.M. **p < 0.01 ***p < 0.001 between indicated groups using unpaired Student’s t-test. (B) qRT-PCR analysis of MMP mRNA expression in cultured BT116 cells. mRNA was collected at indicated time points after stimulation with TWEAK (10 ng/ml) or TNFα (10 ng/ml). Data represent average mRNA expression relative to GAPDH mRNA and normalized to untreated cells at t = 0 (n = 3 replicates) ± S.D. (C) Western blot of whole-cell lysates from cultured BT25, BT116, and U87 cells transduced with shRNA targeting p100/p52 (NFKB2) or a scrambled control (Control). Lysates were collected at 48 h after stimulation with 10 ng/ml TWEAK or vehicle control. White arrowhead indicates p52 band. Black arrowhead indicates non-specific band. (D) qRT-PCR analysis of MMP9 mRNA expression from cell lines in Figure 4C. mRNA was collected at 48 h after stimulation with 10 ng/ml TWEAK or vehicle control. Data represent average MMP9 mRNA expression relative to RPLP0 mRNA and normalized to untreated control (n = 3 replicates) ± S.D. ***p < 0.001 between indicated groups determined by unpaired Student’s t-test for each cell line.