Figure 9.

1,25(OH)₂D₃ down-regulates bic expression by blocking NF-κB activation. (A) Illustration of the putative κB cis-DNA element and ChIP primers within the first intron of the bic gene; (B and C) ChIP assays. LPS promotes p65 binding to the intronic κB site and 1,25(OH)₂D₃ blocks p65 binding in RAW264.7 cells. The DNA fragment precipitated by anti-p65 antibodies was assessed by regular PCR (B) and qPCR (C). *** P<0.001 vs. the rest. (D) EMSA. ³²P-labelled canonical κB probe (Con, lane 1) or bic intronic κB probe (bic, lane 3) was incubated with in vitro translated p65 and p50. ³²P-labelled bic intronic κB probe and NF-κB reaction was carried out in the presence of excess amount of unlabeled bic κB probe (lanes 4 and 5), mutant bic κB probe (lanes 6 and 7) or canonical bic κB probe (lanes 8 and 9). (E) Illustration of bic luciferase reporter constructs with WT and mutant intronic κB sequences. (F) Luciferase reporter assays. RAW264.7 cells were transfected with WT or mutant bic luciferase reporter plasmid followed by treatment with saline control (Cont) or LPS in the presence or absence of 1,25(OH)₂D₃. *** P<0.001 vs. Cont; ### P<0.001 vs. LPS; n=3. (G) IKK kinase assays and IKKβ protein levels in RAW264.7 cells treated with LPS in the presence of ethanol or 1,25(OH)₂D₃; (H) Immunostaining with anti-p65 antibodies of RAW264.7 cells treated with saline control (Cont) or LPS in the presence of ethanol (EtOH) or 1,25(OH)₂D₃.