Gene knock down of TAK-1 and IKK-β impairs TNF-induced stem cell proliferation. (A) NSCs were co-transfected with siRNA for GFP and GFP expression vector to monitor the efficacy of the knock down strategy (upper panel). After fixation, all cells were stained for TAK-1 and IKK-β and counterstained for DNA with DAPI (blue). Note the expression of TAK-1 (red) and IKK-β in cells transfected with siRNA for GFP (green). In contrast, cells co-transfected with siRNA for TAK-1 (red) and GFP expression vector, or with anti IKK-β siRNA and GFP, expressed minimal levels of knocked down proteins (see arrowheads). Note that non-transfected, GFP-negative cells showed unaffected TAK-1 expression (marked by arrowhead). Bars represent 200 μm. Immunocytochemical results were confirmed by western blotting. Note also the unaffected expression of TAK-1 and IKK-β in control (anti GFP siRNA transfected) cells. (B) Western blot analysis of gene knock down. (C) Quantification of proliferation in siRNA transfected cells. After dissociation of transfected neurospheres, the cell number was determined as described in Materials and Methods. Note the stronger negative effect of IKK-β knock down compared to TAK-1 silencing. (D) Reporter gene assay showed down-regulation of NF-κB activity after gene knock down. NF-κB activity was most markedly inhibited after IKK-β knock down. (E) Analysis of cyclin D1 expression in cells transfected with siRNAs. Note that the most prominent effect was observed in IKK-β siRNA-transfected cells after TNF treatment. (F) Activation of caspase 3 in cells transfected with IKK-β and TAK-1 siRNA. Note the strongest effect of IKK-β knock down after TNF stimulation.