Figure 3.

1,25(OH)₂D₃ blocks MLCK activation by targeting NF-κB activation. (A) Western blot analyses of HCT-116 cells treated or untreated with TNF-α ± BAY 11-7082 as indicated. (B) Transepithelial resistance (TER) of HCT116 cell monolayers under different treatments as indicated for 6 hours. *** P<0.001 vs. the rest. (C) HCT116 cells were transfected with empty vector or IKKβ-expressing plasmid, followed by 1,25(OH)₂D₃ or vehicle treatment as indicated. Cell lysates were analyzed by Western blotting. (D) HCT116 cells were transfected with increasing amount of IKKβ and then treated with 1,25(OH)₂D₃ as shown. Cell lysates were analyzed by Western blotting. (E) Schematic illustration of human MLCK gene promoter containing three putative κB sites (κB-1, κB-2 and κB-3) at −136, −1451 and −1584 as indicated. The PCR primer locations were also indicated. (F) ChIP assays using anti-p65 antibodies for HCT116 cells pre-treated with 1,25(OH)₂D₃ or vehicle followed by 4-hour TNF-α or saline treatment as indicated. The results for κB-1, κB-2 and κB-3 sites are shown. Similar results were seen for 10-hour treatment. **P<0.01 vs. the rest.