Figure 2
NIK expression is upregulated in highly invasive GBM cells and promotes collective invasion. (A) Live-cell confocal microscopy was utilized to visualize the RFP reporter under the NIK promoter (pNIK-RFP) (red) under unstimulated conditions (NT). The monolayer (0 h) was pseudocolored white and overlaid to the reference invasion distance at 24 h and 48 h. (B) Graph representation of RFP intensity after 48 h and distance the cells invaded. Pearson’s correlation between RFP intensity and distance is r = 0.788 and p = 0.008, ***p ≤ 0.001. (C) Invasion of BT116 pNIK-RFP cells labeled with DiO (green) with no treatment (NT) or treated with 10 ng/mL TWEAK. (D) BT116 pNIK-RFP cells were used for a spheroid invasion assay. Spheroids were embedded in three-dimensional collagen matrix and either left untreated (NT) or treated with TWEAK (10 ng/mL). Spheres were allowed to invade for 72 h. Confocal microscopy was used to image spheroids, labeled DiO (green), RFP reporter (red), and DAPI (blue). (E) Outline of BT116 pNIK-RFP (red) spheroids embedded in three-dimensional collagen for 0–72 h, either untreated (NT) or treated with TWEAK (TW) for 48 h. (F) The ImageJ particle analysis function was used to quantify the number of cells invading into the three-dimensional collagen matrix at various time points. Data represented as mean±SEM, two-way ANOVA. (G) ImageJ was used to measure the radius of the spheroids at 0 h, 24 h, and 48 h. Data represented as mean±SEM. (H) Three-dimensional projection image of BT116 pNIK-RFP (Red) spheroids embedded in collagen and treated with TWEAK (TW) for 72 h. Cells were labeled with DiO (green) and DAPI (blue). White arrowheads with enhanced images of cells undergoing collective invasion. Spheroid invasion assays were conducted in n > 3 either untreated or treated with 10 ng/mL TWEAK.
