buffer containing 1% NP40, 0.1 M Tris-HCl, 0.15 M NaCl, 5 mM EDTA and protease inhibitors (1 mM ZnCl2, 50 mM Na2MoO4, 10 mM NaF, 0.1 mM NaVO3, 1 mM PMSF, 1 mM DTT, 16 complete EDTA-free protease inhibitor (Roche, Mannheim, Germany). Cell lysates were collected at 13 0006 g for 10 minutes at 4uC. Proteins were fractionated on NuPAGEH NovexH Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred onto a nitrocellulose membrane using the iBlotH system (Invitro- gen). The membrane was blocked in 5% non-fat dry milk in TBS (10 mM Tris-HCl, pH 7.7, 150 mM NaCl) with 0.1% Tween 20 (TTBS) at room temperature for 1 hour, incubated with the indicated primary antibodies overnight at 4uC, washed in TTBS and incubated for 1 hour at room temperature with the corresponding secondary HRP-conjugated antibodies (Amersham Biosciences) in 5% non-fat dry milk in TTBS. Proteins were visualized using ECL PlusTM Chemiluminescent Detection System. Primary antibodies used were: anti-EZH2 (AC22; #3147, Cell Signaling Technology) and anti-Actin (I-19; sc- 1616, Santa Cruz Biotechnology). Resazurin assay RPMI 8226 and U-266-1984 cells were incubated in round bottomed (RPMI 8226) or flat bottomed (U-266-1984) 96-well plates with different concentrations of LBH589 or DZNep. At the indicated time points, 10% AlamarBlue was added to the wells, followed by incubation for 1–3 hours at 37uC in a humidified 5% CO2 in-air atmosphere. Wallac VICTOR Multilabel Counter (Wallac, Turku, Finland) was used for the fluorescence analysis. Resazurin was excited at 530 nm and emitted light was measured at 590 nm. Mean was calculated from triplicate wells and subtracted from mean of blank wells resulting in DFluorescence. The relative number of viable cells was expressed as percentage of untreated cells and calculated as 1006 DFluorescence (treated cells)/DFluorescence (untreated cells). Apoptosis assay RPMI 8226 and U-266-1984 were cultured in 6-well plates for 48 and 72 hours in the presence of LBH589 or DZNep. Apoptosis was quantified by Annexin V (AV)-fluorescein isothocyanate (FITC) and PI staining using TACS Annexin V-FITC Apoptosis Kit (R&D Systems, Gaithersburg, MD, USA). Samples were treated according to manufacturer’s recommendations and analyzed by flow cytometry (FACScan), presenting apoptotic cells as Annexin V-positive/PI-negative cells and necrotic cells as Annexin V-positive/PI-positive cells. Animals C57BL/KaLwRij mice were purchased from Harlan CPB (Horst, The Netherlands). Mice were used at 6 to 10 weeks of age. 5T33MMvv cells At day 0 C57BL/KalwRij mice were injected with 0.56106 5T33MM cells. After 14 days mice were either assigned to a treatment group receiving 10 mg/kg LBH589 (n = 4; daily i.p. injection) or to a vehicle group receiving 0.9% NaCl solution (n = 4; daily i.p. injection). After 5 days of treatment mice were sacrificed and the BM harvested after flushing out of the femurs and tibiae and crushing out of the vertebrae. The BM cells were suspended in serum-free medium (RPMI 1640; Cambrex, Europe), supplemented with penicillin-streptomycin, glutamine, and minimal essential medium (MEM) nonessential amino acid (NEAA)–pyruvate (Gibco, Life Technologies) and purified by Lympholyte M (Cedarlane, Hornby, ON, Canada) gradient Polycomb Silencing in MM centrifugation at 1000 g for 20 minutes, generating enriched 5T33MM cells. For further enrichment, a negative selection for CD11b positive cells was performed using a MidiMACS magnetic cell separator, LD separation columns and CD11b magnetic microbeads (all from Miltenyi Biotec, Bergisch Gladbach, Germany). 5T33MMvv cells at a concentration of 107 cells per 90 ml MACS buffer (phosphate buffered saline containing 0.5% bovine serum albumin and 2 nM EDTA, pH 7.2) and 10 ml CD11b Microbeads were incubated at 4uC for 15 min. The 5T33MMvv-microbeads mixture was then loaded onto a separation column placed on the magnetic cell separator. After the flow through, the column was washed three times using 500 ml of MACS buffer and the eluted cell fraction was collected for further experiments. Assessment of the tumor burden in vivo The 5T33MM cells were injected in C57BL/KalwRij mice as described above. At day 0 mice were either assigned to a treatment group receiving 10 mg/kg LBH589 (n = 10; daily i.p. injection), to a vehicle group receiving 0.9% NaCl solution (n = 10; daily i.p. injection) or to an untreated, disease-free group (n = 10) as a control group. The animals were sacrificed when they showed signs of morbidity (paralysis). Tumor load was analyzed by means of serum paraprotein concentration and BM plasmocytosis: the first was quantified by electrophoresis and assessment of total protein, the latter by quantification of May Gr u¨nwald-Giemsa staining of BM cytospin samples. Survival analysis Survival was studied using Kaplan-Meier analysis. 5T33MM cells were injected as above and at day 0 mice received 10 mg/kg LBH589 (n = 12; daily i.p. injection) or vehicle, 0.9% NaCl solution (n = 12; daily i.p. injection). An untreated disease-free group (n = 12) was used as a control. Each mouse was sacrificed when it showed signs of morbidity (paralysis). Supporting Information Figure S1 The PRC2 components EED, EZH2 and SUZ12 are overexpressed in MM. (A): MM patients (n = 74) compared to plasma cells (n = 37) and tonsillar tissue (n = 37) (Zhan, Hardin et al. 2002). (B): Smoldering Myeloma patients (n = 12) compared to MGUS patients (n = 44) and normal bone marrow (n = 22) (Zhan, Barlogie et al. 2007). (C): MM stage III patients (n = 34) compared to MM stage I (n = 30) and stage II (n = 38) (Agnelli, Fabris et al. 2007). Analysis was performed on normalized expression units, using the built in t-statistics (Rhodes, Yu et al. 2004). Found at: doi:10.1371/journal.pone.0011483.s001 (0.99 MB TIF) Figure S2 The PRC2 targets CIITA, GATA2, CDH6, CXCL12 and ICSBP/IRF8 are underexpressed in MM. Target genes were defined by Bracken et al. (Bracken, Dietrich et al. 2006) in embryonic fibroblasts and their expression pattern examined using Oncomine. (A): MM patients (n = 74) compared to plasma cells (n = 37) and tonsillar tissue (n = 37) (Zhan, Hardin et al. 2002). (B): Smoldering Myeloma patients (n = 12) compared to MGUS patients (n = 44) and normal bone marrow (n = 22) (Zhan, Barlogie et al. 2007). (C): MM stage III patients (n = 34) compared to MM stage I (n = 30) and stage II (n = 38) (Agnelli, Fabris et al. 2007). Analysis was performed on normalized expression units, using the built in t-statistics (Rhodes, Yu et al. 2004). Found at: doi:10.1371/journal.pone.0011483.s002 (1.51 MB TIF) Figure S3 The H3K27-tri-methylated CIITA, GATA2, CDH6, CXCL12 and ICSBP/IRF8 are enriched among genes under- PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11483