Polycomb Silencing in MM Figure 1. H3K27me3 targets are underexpressed in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). The H3K27me3 target genes (n = 2390) were defined by Bracken et al. [19] in embryonic fibroblasts and found to be statistically associated to underexpressed genes in MM and MGUS using Oncomine. (A): The top 10% underexpressed genes [7] (n = 456) in MM patients (n = 74) compared to plasma cells (n = 37) and tonsillar tissue (n = 37) showed significant (p = 2.1361029) overlap with the H3K27me3 target genes. (B): The top 10% underexpressed genes [20] (n = 1907) in MGUS patients (n = 44) compared to normal bone marrow (n = 22) showed significant (p = 5.5610210) overlap with the H3K27me3 target genes. (C): The top 10% underexpressed genes [5] (n = 1242) in MM stage III (n = 34) compared to MM stage I and stage II (n = 68) showed significant (p = 1.3610233) overlap with the H3K27me3 target genes. (D): H3K27me3 target genes expression during MM progression [5]. doi:10.1371/journal.pone.0011483.g001 (RPMI 8226) and 4, 20 and 100 nM (U-266-1984). Depletion of EZH2 protein levels measured by Western blotting analysis was observed in a dose-dependent manner for both drugs in both cell lines (Figure S4A–D). Treatment with 10 mM DZNep for 6, 24, 48 and 72 hours led to a gradual depletion of EZH2 protein in both the RPMI 8226 and the U-266-1984 cells (Figure 4A and 4B). Additionally, the EZH2 protein level was reduced after treatment with 20 nM LBH589 with the strongest reduction observed at the latest time point of treatment, 48 hours for U-266-1984, and 72 hours for RPMI 8226 (Figure 4C and 4D). PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11483