Figure 5
β-Catenin and NF-κB co-mediate TLR3 activation-induced CSC phenotypes. (a) qPCR analysis of target genes of β-catenin pathway (Axin2 and CylinD1) in SUM190 after treatment with 1 μg/ml of poly(I:C) for 4 days. Data represent the average±S.D., n=5; *P<0.05. (b) Western blot analysis of nuclear translocation of active β-catenin in SUM190 cells after treatment with poly(I:C) (1 μg/ml ) for 4 days. α-tubulin and Lamin A/C: internal loading controls for cytoplasmic (Cyto) and nuclear (Nu) proteins. (c) qPCR analysis of the indicated stem markers. SUM190 cells were pretreated for 4 h with Bay 11-7821 (5 μM, an NF-κB inhibitor) and BC21 (7.5 μM, a β-catenin/TCF4 inhibitor) either alone or in combination, followed by stimulation with poly(I:C) for 4 days in the presence or absence of the above inhibitors. Data represent the average±S.D., n=3; *P<0.05. (d) Inhibition of both NF-κB and β-catenin pathways completely blocks poly(I:C)-induced expression of CD44 and c-Myc (western blot). SUM190 cells were treated in the same way as those described in (c). (e) SUM190 cells were transfected with siRNA oligos against NF-κB p65 (p65), β-catenin or non-targeting oligos for 24 h, and then treated with 1 μg/ml of poly(I:C) for 3 days. Immunoblotting shows an almost complete knockdown of NF-κB and β-catenin expression after co-transfection of NF-κB p65 and β-catenin siRNA. (f) Knockdown of both NF-κB (p65) and β-catenin abrogates an increase in CD44high/CD24−/low cell population following poly(I:C) treatment. Data represent the average±S.D., n=3; *P<0.05
