The effect of TNF on NSCs. (A) TNF activates NF-κB in NSCs as measured by reporter gene activity and nuclear translocation of the p65 subunit. Transfection efficacy was measured with CMV-driven GFP expression plasmids (upper panel). The left image depicts a phase contrast image of transfected cells. The middle image shows GFP expression. Both images are merged in the right image. Bar depicts 200 μm. Flow cytometry was used to quantify the transfected cells. Control cells (untransfected) are shown in white. Transfected cells are shown in grey. Note the high efficacy of transfection (about 60%). (B) NSCs were transfected using the optimized conditions shown in (A). Induction of NF-κB by TNF was more than ten-fold. Basal κB-dependent reporter gene expression, as measured by bioluminescence under control conditions, was set to 1. (C) TNF-induced nuclear localization of the transactivating NF-κB subunit p65. Nuclei were stained with SYTOX (green). Activity-specific anti-p65 immunoreactivity is depicted in red (middle panel). Merged images are shown on the right. Note the basal NF-κB activity in unstimulated cells (upper panel). Bar depicts 5 μm. (D) Representative fields of cells were analyzed to determine the mean p65 fluorescence in the nucleus. Note that TNF strongly increases nuclear p65 compared to untreated controls. (E) TNF treatment elevates cyclin D1 expression in NSCs. Cold-shock synchronized neurospheres were collected on glass slides by cytospin centrifugation, fixed and stained for cyclin D1. DNA was stained using SYTOX (green). Note the higher cyclin D1 expression in TNF-treated cells (lower panels) compared to controls (upper panels). (F) Quantification of cyclin D1 expression. TNF-treated neurospheres show significantly higher numbers of cyclin D1-positive cells than untreated neurospheres (*** P < 0.001). Bars, 20 μm.