FIGURE 7.

p16, Sp1, and CDK4 proteins form a heterocomplex that binds the miR-141 and miR-146b-5p promoters. A, whole cell extracts were prepared from HFSN1 cells and immunoprecipitated (IP) with anti-p16, anti-Sp1, or anti-CDK4 antibodies (mouse IgG was used as control), and immunoblotting was performed using the indicated antibodies. B, EMSA. Supershift assay was performed using nuclear extracts from the indicated cells that were preincubated with the indicated antibodies (1 μg) for an hour before adding the biotin-labeled oligonucleotides of the Sp1 consensus-binding site of the miR-146b-5p promoter. C, EMSA. Pure proteins were used in the indicated combinations and biotin-labeled oligonucleotides of the Sp1 consensus binding sites of the miR-141 and miR-146b-5p promoters. The numbers indicate the amount of proteins in nanograms. D, ChIP assay. Chromatin was purified from the indicated cells and then immunoprecipitated using anti-p16 or anti-CDK4 antibodies. miR-141 and miR-146b-5p promoters were amplified by PCR (upper panel) and qPCR (lower panel), and their abundances were plotted relative to the input. E, total proteins and RNA were prepared from HFSN1 cells expressing either control-shRNA or CDK4-shRNA and were used to assess the level of CDK4 by immunoblotting and the level of miR-141 and miR-146b-5p by qRT-PCR. Error bars represent means ± S.D.