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Results: 6

1.
Figure 4

Figure 4. DZNep and LBH589 deplete EZH2 protein level in MM cell lines.. From: Polycomb Target Genes Are Silenced in Multiple Myeloma.

(A) and (B): The RPMI 8226 cells (A) and the U-266-1984 cells (B) were treated with DZNep (10 µM) for 6, 24, 48 and 72 hours. (C) and (D): The RPMI 8226 cells (C) and the U-266-1984 cells (D) were treated with LBH589 (20 nM) for 6, 24, 48 and 72 (RPMI 8226) or 6, 24 and 48 hours (U-266-1984). Western blot was performed using a specific antibody against EZH2. Actin was used to control for equal loading.

Antonia Kalushkova, et al. PLoS One. 2010;5(7):e11483.
2.
Figure 3

Figure 3. The expression of H3K27-tri-methylated genes is reactivated by DZNep and LBH589 in MM cell lines.. From: Polycomb Target Genes Are Silenced in Multiple Myeloma.

The RPMI 8226 cells (A) and (C), and the U-266-1984 cells (B) and (D) were treated with DZNep (10 µM) or LBH589 (20 nM) for 6, 24, 48 and 72 hours. Fold difference in expression was calculated relative to the untreated cells. GAPDH was used as a reference gene. Error bars represent SD from three independent biological experiments.

Antonia Kalushkova, et al. PLoS One. 2010;5(7):e11483.
3.
Figure 5

Figure 5. DZNep and LBH589 reduce growth and induce apoptosis in MM cell lines.. From: Polycomb Target Genes Are Silenced in Multiple Myeloma.

(A) and (B): U-266-1984 and RPMI 8226 cells were treated for 6, 24, 48 and 72 hours with DZNep (10 µM) or LBH589 (20 nM) followed by a resazurin assay. At least 2 independent experiments were performed in triplicates, data is presented as mean percentage of control ±SD. (C) and (D): U-266-1984 and RPMI 8226 were treated with DZNep (10 µM) and LBH589 (20 nM) for 72 hours followed by AV/PI staining and flow cytometry analysis. At least 2 independent experiments per cell line were performed; data is presented as mean percentage apoptotic cells ±SD.

Antonia Kalushkova, et al. PLoS One. 2010;5(7):e11483.
4.
Figure 2

Figure 2. Polycomb target genes are enriched for H3K27-tri-methylation in MM patient cells and MM cell lines.. From: Polycomb Target Genes Are Silenced in Multiple Myeloma.

Chromatin immunoprecipitation (ChIP) assay using (A)–(D): antibody against H3K27-tri-methylation in purified CD138+ cells from MM patients; (E) and (F): antibodies against H3K27-tri-methylation and H3K9-acetylation in RPMI 8226 and U-266-1984 cells. The RPL30 and GAPDH genes were used as a negative control for H3K27-tri-methylation and a positive control for H3K9-acetylation. The CIITA, CXCL12, GATA2, CDH6 and ICSBP genes were selected from the integrative genomics profile. * The INK4A gene was selected from the literature. Immunoprecipitated DNA was analyzed using real-time qPCR. Specific signal was calculated as fold change between signal and background (IgG) noise normalized to percent input. (E) and (F): Error bars represent SD from three independent biological experiments.

Antonia Kalushkova, et al. PLoS One. 2010;5(7):e11483.
5.
Figure 1

Figure 1. H3K27me3 targets are underexpressed in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS).. From: Polycomb Target Genes Are Silenced in Multiple Myeloma.

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 = 2.13×10−9) 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 = 5.5×10−10) 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 = 1.3×10−33) overlap with the H3K27me3 target genes. (D): H3K27me3 target genes expression during MM progression [5].

Antonia Kalushkova, et al. PLoS One. 2010;5(7):e11483.
6.
Figure 6

Figure 6. LBH589 upregulates gene expression, reduces tumor load and increases survival in the 5T33MM mice model.. From: Polycomb Target Genes Are Silenced in Multiple Myeloma.

(A) and (B): At day 0 C57BL/KalwRij mice were injected with 0.5×106 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, BM harvested and a negative selection for CD11b positive cells performed. CIITA and CXCL12 fold difference in gene expression was calculated relative to the vehicle by Q-RT-PCR. GAPDH was used as a reference gene. The error bars represent SEM from two independent Q-RT-PCR runs. Each group contained one mouse, excluding the vehicle non-depleted, which contained two mice. (C) and (D): Mice were treated as described above, with the difference that 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 at day 0. The animals were sacrificed when the vehicle group showed signs of morbidity. Tumor load as determined by serum electrophoresis (C) and tumor load as determined by May Grünwald-Giemsa staining of BM cytospin samples (D) Mean values ± SD for groups of 10 mice are shown (*p<0.0001). (E) Survival was studied using the Kaplan-Meier analysis method. Mice were treated as described above with the exception that treatment continued until each animal showed signs of morbidity. Each group contained 12 mice (p<0.0003).

Antonia Kalushkova, et al. PLoS One. 2010;5(7):e11483.

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