The Proceedings of the Eighth International Conference on Creationism (2018)

competitively inhibited with an unlabeled, unmethylated version of the PRE probe, but the unmethylated probe was more easily competed off, suggesting sequence-specific binding of both probes and the higher-affinity binding of the methylated probe. The irrelevant OCT4 probe did not inhibit protein-binding of the labeled PRE probes to the same extent, further indicating that the interaction between proteins and the PRE probes was sequence- specific. Protein-binding kinetics demonstrated preferential binding of PR protein to methylated DNA. In this experiment, 50 fmoles of each probe was allowed to react with increasing concentrations of nuclear extract. As shown in Fig. 4 D, the methylated probe bound low concentrations of proteins more tightly, while the unmethylated probe only bound proteins when the concentrations of nuclear extract were high. A plot of bound fractions of the fluorophores against concentrations of nuclear extract revealed distinct binding kinetics of the two probes (Fig. 4E). The methylated probe approached a second-order polynomial kinetics and was saturated within the range of the experiment, while the unmethylated probe demonstrated a third order polynomial kinetics and was not saturated within the range of the experiment. To confirm that the shifted band contained progesterone receptors, a mouse monoclonal antibody that binds both isoformA and isoform B of progesterone receptors was used in supershifting experiments. The antibody consistently supershifted the DNA-protein complex, as seen in Fig. 4F. Significantly, a rabbit monoclonal antibody against OCT4 also supershifted the complex, indicating that OCT4 was also in the complex. Liu and Nguyen ◀ Endogenous Retroviruses ▶ 2018 ICC 196 Figure 3. OCT4 mediates the effect of progesterone in T47D cells. A. Effect of OCT4-specific siRNA and random duplex RNA on the expression of the env gene of HERVK10. Cells were treated with female sex hormones in the presence of siRNA against OCT4 or random duplex RNA. Quantitative RT-PCR was carried out in a manner similar to that in Fig. 1. * indicates significant difference compared with random RNA. B. Effect of female sex hormones on the expression of OCT4 in T47D cells. Cells were treated with sex hormones and antagonists as described in Fig. 1. OCT4 transcript was quantified with quantitative RT-PCR. *denotes significant difference compared with untreated control cells. D. Effect of decitabine treatment and OCT4 overexpression on the expression of HERVK10 env in primary mammary epithelial cells. Decitabine was used at 10 µM for four days. pcDNA_OCT4 was transfected into cells 48 hours prior to RNA harvest. Treatment with female sex hormones was essentially the same as with T47D cells, except progesterone was used at 100 nM. Average of two independent experiments.