Figure 4. TLR2 participates in BCG-induced activation of MEK pathway for AMP release in bladder cancer cells.

(A) Western blotting analyses demonstrating the reduction of Toll-like Receptor 2 (TLR2) or TLR4 protein expression in T24 cells transfected with two shRNA-expressing plasmids targeting TLR2 or TLR4 sequences (shTLR2#1, shTLR2#2, shTLR4#1 and shTLR4#2, respectively; upper panels). Non-specific scrambled shRNA was used as control. shRNA TLR2 or TLR4 knockdown cells were selected with puromycin, and infected with BCG (10 or 30 MOI for 48 h; lower panels). Phospho ERK expression was detected by Western blotting. (B) Stable clones of TLR2- or TLR4-knockdown T24 cells were treated with BCG (10 MOI for 8 h) and analyzed for expression of phosphorylated c-Jun. Phospho-Jun/total Jun expression ratios were calculated by densitometric analysis, and indicated under each lane. Expression ratios were normalized to the untreated group. (C) StableshTLR2 or shTLR4 T24 bladder cancer cells were infected with BCG (10 MOI for 8 h), followed by ELISA of antimicrobial peptides (HBD-2, HBD-3, and CAMP) in the culture supernatant. Data are mean ± SD (n=3 per group). * P<0.05 and ** P<0.01 Student's T-test. (D) ChIP assays using anti-c-Jun antibody to detect physical interactions between c-Jun and HBD2, HBD-3 or CAMP. TLR2-or TLR4-knockdown T24 cells were infected with BCG (10 MOI for 8 h) and cross-linked with 10% formaldehyde. Expressions of AMPs were calculated as the ratio of c-Jun to Input DNA expression using densitometric analysis. All expression ratios were normalized to the untreated group.