Figure 5. MYCN expression is directly regulated by BRD4 and repressed by treatment with I-BET726.

(a) Left: Concentration response curve for MYCN RNA expression following 24 hour treatment with I-BET726 in the CHP-212 cell line. Data was normalized to GAPDH and is presented as expression relative to DMSO-treated controls. Data presented as the average of two independent curves from a single experiment, and is representative of data from three independent biological replicates. Right: Table of IC₅₀ and percent inhibition values for MYCN suppression following 24 hour treatment with I-BET726 in the indicated cell lines. (b) BRD4 ChIP in the non-MYCN-amplified cell line SK–N–SH. Binding of BRD4 to the MYCN promoter or to an intergenic region on Chromosome 12 following treatment with vehicle or 1µM I-BET726 for six hours. Data is presented as fold enrichment over signal generated from IgG control immunoprecipitations. Data shown is from a single experiment representative of typical results. (c) BRD4 ChIP data at the MYCN promoter, presented as percent of vehicle control signal in the non-MYCN-amplified cell line SK–N–SH (left) and the MYCN-amplified cell line CHP-212 (right). SK–N–SH data represents the mean value ± standard deviation for three independent biological replicates. Asterisk indicates statistical significance as measured by t-test (p= 0.005). CHP-212 data represents the mean value ± standard deviation for two independent biological replicates. (d) Western blot analysis of N-Myc expression in the MYCN-amplified cell lines CHP-212 and IMR32 following 24 or 48 hour treatment with vehicle or 1µM I-BET726. Actin expression included as a loading control. (e) gIC₅₀ values obtained from CHP-212 cells overexpressing GFP or MYCN following treatment with I-BET726 in a 6 day growth-death assay. Data represents the mean value ± standard deviation from four independent experiments. (f) Concentration response curves for GFP or MYCN–overexpressing CHP-212 cells from a 6 day growth-death assay. Horizontal line indicates T₀ measurement (normalized to 100%). Data shown was from a single experiment representative of typical results. (g) Left: Histograms generated from cell cycle analysis in GFP- or MYCN-overexpressing CHP-212 cells following 4 days treatment with 5 µM I-BET726. Right: Percentage of cells in subs G1, G1, S, and G2 phases from the cell cycle experiment.