FIGURE 6.

IL-1β enhances the endogenous ELF3 binding to the proximal MMP13 promoter. A, after overnight incubation in serum-free medium, T/C-28a2 cells were incubated with 1 ng/ml IL-1β for 2 h. After stimulation, the chromatin was cross-linked and enzymatically sheared, and after reverse cross-linking of the DNA-protein complexes, the precleared lysates were incubated with antibodies against ELF3 (+) or normal IgG (−) overnight at 4 °C. The human MMP13 promoter region was PCR-amplified using primers spanning from −157 to −38 bp, and the PCR products were resolved on a 2.5% agarose gel. B, ELF3 protein levels were analyzed by Western blotting using cell lysates prepared from T/C-28a2 cells stimulated with vehicle or 1 ng/ml IL-1β for 2 h. C, T/C-28a2 cells were transfected with 50 nm siRNA oligonucleotides against ELF3 (siELF3) or non-targeting siRNA (siCONTROL). At 72 h post-transfection, cells were stimulated with 1 ng/ml IL-1β for 6 h. Total RNA was isolated, and MMP13 mRNA was analyzed by RT-qPCR. Each value was normalized to GAPDH in the same sample and shown as mean ± S.E. *, p < 0.05. IP, immunoprecipitation.