Figure 4. CHR-3996 treatment increases levels of acetylated histone H3K9 but does not affect levels of ubiquitinated proteins, acetylated alpha-tubulin, or inhibit proteasome function.

A. H929 (i) and RPMI-8226 (ii) cells were treated with CHR-3996 (250 and 100 nM respectively) and primary patient CD138⁺ plasma cells (iii) were treated with either 50 (left panel) or 100 nM (right panel) CHR-3996 over a time-course of 48 hours. Following cell lysis histones were released from DNA by overnight extraction with 0.2M HCl and immunoblotting performed to detect acetylated H3K9. B. H929 (i) and RPMI-8226 (ii) cells were treated with CHR-3996 (250 and 100 nM respectively) or bortezomib (8nM) for 8, 24, and 48 hours. Following cell lysis immunoblotting was performed to detect ubiquitin, acetylated a-tubulin, and actin. Crtl represents untreated cells. C. H929 and RPMI-8226 cells were treated with CHR-3996 (250 and 100 nM respectively) or bortezomib (4 nM). Following cell lysis 25 μg of protein was added to Suc-Leu-Leu-Val-Tyr-AMC, substrate for the chymotryptic activity of the proteasome, and the fluoresence read from each well every 120 seconds for 48 repeats. The activity was calculated from the rate of fluorescence detected in the linear phase of the reaction and shown as a percentage of untreated cells.