Figure 6.

Panel A. Induction of global H3K4me2 following treatment with a 10 μM concentration of compounds 6b, 6c or 6d. Calu-6 cells were seeded at a density of 400,000 per T25 flask. Upon 60% confluency, the cells were treated with 10 μM of 6b, 6c, or 6d for 24h or 48h. 30 μg of nuclear extract was used for Western blot analysis. Non-treated cells are denoted as NT. Gel bands were quantitated using the Odyssey software, and H3K4me2 bands were normalized to total histone H3 bands. The graphical data are the means ± S.E. from four experiments. Student’s t tests were used to determine statistical significance. *p<0.1 and **p<0.05. Panel B. 5 μg of full-length LSD1 purified protein was incubated with 5 μg of bulk histones in the presence or absence of 5, 10, or 20 μM LSD1 inhibitor. The reaction was incubated at 37°C for 3h. Western blot analysis was performed and H3K4me2 levels were determined using the Odyssey Software program. All of the data were normalized to the histone only control and are shown as a percent of H3K4me2 in relation to the histone only condition. Data points in Panel B represent quantitation of single Western blots from a representative experiment that was repeated 4 times with similar results.