Figure 3

LPS induced activation of p38 MAPK and NF-κB pathways through TLR4 in OSCC cell lines. A. LPS induced p38 MAPK phosphorylation and I-κBa degeneration for indicated time points in OSCC cell lines. B. HIOEC and OSCC cells plated overnight were stimulated with LPS (1 μg/ml) for 8 h and then stained as described in Materials and Methods. a to c, control cells were untreated; a and d, nuclei are stained blue; b and e, the p65 subunit of NF-κB is stained green; c, an overlay of a and b; f, an overlay of d and e. C. Mean ± SD of the percentage of cells with p65 translocation. 200 cells were randomly counted. Results are representative of three independent experiments performed with each cell line (*p < 0.05, compared with control). D. Mean fluorescence intensity (MFI) of nuclear p65 expression was determined in OSCC cell lines. Results are expressed as mean ± SD of MFI for three independent experiments performed with each cell line (*p < 0.05, compared with control). E. Determination of the LPS-activated, NF-κB-dependent transcriptional activity in CAL-27 cells. The cells were transiently cotransfected with pNF-κB-Luc and pRenilla. Then after 24 h, cells were either left untreated or stimulated with 1 μg/ml of LPS for various times. Luciferase activity was assessed in the cells (*p < 0.05, compared with control). F. TLR4 was effectively silenced as determined by Western blot and TLR4 siRNA suppressed the activation of p38 MAPK and NF-κB pathways in OSCC cell lines treated with LPS. G. NF-κB-dependent transcriptional activity was inhibited by TLR4 siRNA in CAL-27 cells. CAL-27 cells were transiently transfected with NC siRNA and TLR4 siRNA, then cotransfected with pNF-κB-Luc and pRenilla. 24 h later, the cells were either left untreated or stimulated with 1 μg/ml of LPS for various time. Luciferase activity was assessed in the cells stimulated with or without LPS. (*p < 0.05, compared with NC siRNA).