Fig. 4: Kinetics of EC-driven transcriptional repression of the MYC locus.

A Dose-response analysis of pharmacodynamic transcriptional repression of the MYC locus at the bulk level using median fluorescence intensity (FI) or at the single cell level by categorizing cells as “expressing” or “downregulated” reflected these differential kinetics as steeper Hill coefficients in MYC-EC treated conditions. Flow cytometric analysis of K-562 MYC/d2GFP cells treated with MYC-EC or JQ1 using four-parameter logistic regression shown. Median FI in MYC-EC treated cells (orange) reaches a lower best-fit bottom of 1800 compared to JQ1 treated cells (5549; black) with a steeper HillSlope (−1.724 vs. −0.7816; P = 0.0004, F-test). Using GFP as a gate to classify cells as transcriptionally active or repressed at the MYC locus, MYC-EC treatment (teal) results in a steeper HillSlope compared to JQ1 treatment (purple; −1.406 vs. −0.0398; P = 0.0008, F-test). Plotted values represent the mean of three biological replicates ± standard deviation (SD). FACS gating strategy depicted in Supplementary Fig. 3B and C. B Amplicon methylation sequencing was used to assess MYC promoter methylation in K-562 MYC/d2GFP cells treated with 0.625 µg/ml MYC-EC for 44 h. The unsorted cells showed an average CpG methylation across the amplicon of 64% (black), whereas 96% of CpGs in the amplicon were methylated after sorting for functionally delivered cells (orange, GFP-/tdTomato +). 1% of CpGs were methylated in the amplicon in untransfected, MYC On cells (GFP + /tdTomato-). Plotted values represent mean CpG methylation across the amplicon ± SD from 3 technical replicates (****P < 0.0001, one-way ANOVA). FACS gating strategy depicted in Supplementary Fig. 4. Source data are provided as a Source Data file.