Figure 4

NF-κB signal is not exclusively responsible for enhanced CSC phenotypes after TLR3 activation. (a) SUM190, SUM149, BT483, and Cama-1 cells were treated with 1 μg/ml poly(I:C) for 4 days. Nuclear extracts (Nu) and cytoplasmic extracts (Cyto) were immunoblotted to examine nuclear translocation of NF-κB p65. α-tubulin and Lamin A/C: internal loading controls for cytoplasmic and nuclear proteins, respectively. (b) qPCR analysis of target genes of NF-κB pathway: IL8 and IκBα in SUM190, SUM149, BT483, and Cama-1 following 1 μg/ml of poly(I:C) treatment for 4 days. Data represent the average±S.D., n=3; *P<0.05. (c) qPCR analysis of the indicated CSC-associated genes. Cells were treated with NF-κB inhibitor Bay 11-7821 (SUM190:5 μM; SUM149:7.5 μM) or vehicle (control) for 4 h and then co-treated with 1 μg/ml of poly(I:C) or vehicle for 4 days. The expression levels of CSC-associated genes are decreased but not completely abolished after NF-κB inhibition. Data represent the average±S.D., n=3; *P<0.05. (d) Flow-cytometry analysis of SUM190 CD44^high/CD24^−/low sub-population after the same treatment as described in (c). Data represent the average±S.D., n =3; *P<0.05; **P<0.01