Fig. 6.

Effect of RA on binding of TLR4-MD-2 complex in human colon cancer cells exposed to inflammatory microenvironments. (A) MTT assay was used to detect the cytotoxic effect of RA on HCT116 and HT29 cells. Experiments were performed in triplicate in a parallel manner, and the values were represented as means ± standard deviations. (C-E) Every group was treated with CM. In the conditioned culture system, the ratio of the colon cancer cell media to the THP-1 supernatant was 1:5. The vehicle and LPS 20 ug/mL groups were treated with CM from the nonstimulated THP-1 cell, and received no treatment or were treated with LPS 20 ug/mL. The cells exposed to CM from PMA-activated THP-1 cells received no treatment or were treated with 25, 50 μM RA or 20 μg/mL LPS. Then, the cells were further incubated for 5 min. (C-D) Immunofluorescence staining of TLR4 (green) and 4’6-diamidino-2-phenylindole (DAPI; blue) in CM-treated (C) HCT116 cells and (D) HT29 cells. LPS was used as a positive control to evaluate the effects of CM on HT29 and HCT116 cells via TLR-4 mediated NF-κB/STAT3 pathways. (E) Binding activity of TLR4 and MD-2 complexes was analyzed using HT 29 and HCT116 cell lysates using co-immunoprecipitation in triplicate. Values are presented as means ± standard deviations; ^###P < 0.001 vs Veh (Vehicle) group where TLR4 was pulled down from cancer cell; ^⁎⁎P < 0.01 and ^⁎⁎⁎P < 0.001 vs CM group where TLR4 was pulled down from cancer cell; significances between groups were determined by analysis of variance and Dunnett's post hoc test.