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. 2014 Mar 21;9(3):e92793. doi: 10.1371/journal.pone.0092793

Figure 6. Elimination of 3MC-mediated increases in E2F/HDAC1 binding to the E2F responsive element in the promoters of Cdk2/4 and CyclinD3/E1 by simvastatin treatment.

Figure 6

(A) Cells were cultured and treated with 100 nM of 3MC for 1 h after 1 h of simvastatin pretreatment. Nuclear proteins were assayed for E2F binding activity by WT and mut probes in an EMSA assay, as described in Materials and Methods. The term “100xcold” denotes a 100-fold molar excess of unlabeled oligonucleotides relative to the biotin-labeled probe; this was added to the binding assay to compete with the unlabeled oligonucleotides. The mobility of the E2F-E2F responsive element complex is indicated. Representative results of 3 experiments are shown. (B) A ChIP assay was performed in cells that received simvastatin pretreatment for 1 h, or followed by the 3MC challenge for 1 h, as indicated. The DNA associated with HDAC1 was immunoprecipitated with an anti-HDAC1 antibody; thereafter, PCR amplification was used to determine the extent of the association between HDAC1 and the functional E2F-binding sites in the promoters of Cdk2/4 and Cyclin D3/E1. An anti-GAPDH antibody was used as a negative control for the ChIP assays. Representative results of 3 experiments are shown, and data are presented as the mean ± SEM (*P<0.05 vs. the control; # P<0.05 vs. 3MC treatment alone).