Figure 2.
Che-1 sustains histone acetylation. (A) Histone acetylation analysis by mass spectrometry from Kms27 cells transiently transfected with siRNA control (siControl) or siRNA Che-1 (siChe-1). (B) Che-1/H3ac correlation in 37 MM samples evaluated by using western blot (WB) analysis and densitometric analysis. R score, 0.783; P < .0001. (C) Representative WB analysis with the indicated antibodies of total cell extracts from CD138+ purified plasma cells of primary tumors. (D) CD138+ neoplastic cells from patients with symptomatic myeloma were cocultured with stromal cells from the same patient for 24 hours before infecting them with shChe-1 or shControl lentiviral vectors. After 48 hours, total cell extracts from infected MM primary cells were analyzed by WB with the indicated antibodies. (E) Decreased levels of H3K27ac and increased levels of H3K27me3 were found in Kms27 cells transiently transfected with siChe-1 compared with siControl. The bar plot shows the average modulation of histone H3 modifications observed in these experiments (n = 3). P value was calculated by Student t test; *P = .043, **P = .0039. (F) Enrichment density of H3K27ac ChIP-seq significant peaks identified in siControl (blue) and siChe-1 (yellow) samples. Enrichment density was calculated on the relative size of each peak, with 2 replicates per group. Boxplot shows the peak sizes. (G) H3K27ac ChIP-seq signal in siControl (blue) and siChe-1 (yellow) samples at AATF, SCN1A, WDR48 loci. Signal enrichment scale, 0 to 5. (H) Bar plot showing the number of H3K27ac ChIP-seq peaks in common and specific to siControl and siChe-1 samples. (I) Boxplot showing the relative distance of each significant peak to the closest gene in siControl (blue) and siChe-1 (yellow). Distance calculated as absolute value and expressed in kilobase. (J) Kms27 cells transiently transfected with siControl or siChe-1 were subjected to ChIP–quantitative polymerase chain reaction (ChIP-qRT) using anti-H3K27ac antibody (Ab). Data are expressed as percentage of input. Data represent the mean ± standard deviation. P value was calculated by Student t test (n = 3; *P < .023). See also supplemental Figure 2. Mgus, monoclonal gammopathy of undetermined clinical significance.

Che-1 sustains histone acetylation. (A) Histone acetylation analysis by mass spectrometry from Kms27 cells transiently transfected with siRNA control (siControl) or siRNA Che-1 (siChe-1). (B) Che-1/H3ac correlation in 37 MM samples evaluated by using western blot (WB) analysis and densitometric analysis. R score, 0.783; P < .0001. (C) Representative WB analysis with the indicated antibodies of total cell extracts from CD138+ purified plasma cells of primary tumors. (D) CD138+ neoplastic cells from patients with symptomatic myeloma were cocultured with stromal cells from the same patient for 24 hours before infecting them with shChe-1 or shControl lentiviral vectors. After 48 hours, total cell extracts from infected MM primary cells were analyzed by WB with the indicated antibodies. (E) Decreased levels of H3K27ac and increased levels of H3K27me3 were found in Kms27 cells transiently transfected with siChe-1 compared with siControl. The bar plot shows the average modulation of histone H3 modifications observed in these experiments (n = 3). P value was calculated by Student t test; *P = .043, **P = .0039. (F) Enrichment density of H3K27ac ChIP-seq significant peaks identified in siControl (blue) and siChe-1 (yellow) samples. Enrichment density was calculated on the relative size of each peak, with 2 replicates per group. Boxplot shows the peak sizes. (G) H3K27ac ChIP-seq signal in siControl (blue) and siChe-1 (yellow) samples at AATF, SCN1A, WDR48 loci. Signal enrichment scale, 0 to 5. (H) Bar plot showing the number of H3K27ac ChIP-seq peaks in common and specific to siControl and siChe-1 samples. (I) Boxplot showing the relative distance of each significant peak to the closest gene in siControl (blue) and siChe-1 (yellow). Distance calculated as absolute value and expressed in kilobase. (J) Kms27 cells transiently transfected with siControl or siChe-1 were subjected to ChIP–quantitative polymerase chain reaction (ChIP-qRT) using anti-H3K27ac antibody (Ab). Data are expressed as percentage of input. Data represent the mean ± standard deviation. P value was calculated by Student t test (n = 3; *P < .023). See also supplemental Figure 2. Mgus, monoclonal gammopathy of undetermined clinical significance.

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