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Blood, Vol. 114, Issue 3, 651-658, July 16, 2009
Global reduction of the epigenetic H3K79 methylation mark and increased chromosomal instability in CALM-AF10–positive leukemias
Blood Lin et al.
114: 651
Supplemental materials for: Lin et al
Files in this Data Supplement:
- Table S1. The percentages of H3K79 methylation determined by mass spectrometry (PDF, 106 KB)
- Table S2. Primary data of genomic instability assay in T-REx-293 cells stably expressing CALM-AF10ΔOM-LZ or CALM-AF10 (PDF, 261 KB) -
105 and 100 individual chromosome spreads were evaluated, respectively.
- Figure S1. Analysis of histone methylation of the three human monocytic cell lines (JPG, 102 KB)
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(A) Mass spectrometry analysis of H3-K79 methylation in total histone extracts from the three cell lines. (B) Western analysis for histone methylation at various sites. All the antibodies except for α-H3 are specific for dimethylation of the indicated lysine residues.
- Figure S2. The expression of CALM-AF10 does not affect the transcript level of hDOT1L (JPG, 75.8 KB)
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Total RNA was extracted from U937, HL60 and T-REx-293 cells (un-induced and induced for CALM-AF10) followed by RT-PCR assay for hDOT1L expression. The ACTIN expression was used for normalization.
- Figure S3. Transfected AF10 is partially chromatin-bound but CALM-AF10 and hDOT1L are not (JPG, 201 KB)
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(A) Diagram of the cell fractionation procedure. (B) Western detection of transfected AF10, CALM-AF10 and hDOT1L in the cellular fractions. Transfected 293T cells were subjected to cell fractionation. The fractionation efficiency was confirmed by the detection of PCNA in the soluble fraction, histone H3 in chromatin, and lamin B in the matrix. (C) Immunostaining of metaphase chromosomes of transfected 293T cells. Scale bar: 1 µm.
- Figure S4. Global H3-K79 hypomethylation and high frequency of additional cytogenetic aberrations in MLL-AF10-positive patients (JPG, 96 KB)
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(A) H3K79 hypomethylation in leukemia cells with MLL-AF10 fusions. Whole cell extracts were prepared from AML patient samples and analyzed by Western blotting. (B) Increased number of additional cytogenetic aberrations in MLL-AF10 patients compared to patients without H3-K79 hypomethylation (MLL-AF9 and PML-RARA). Secondary: one additional rearrangement to the primary translocation. Complex: two or more additional rearrangements.
- Figure S5. The HOXA5 locus shows local H3K79 hypermethylation in U937 cells compared with HL60 cells (JPG, 107 KB)
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(A) HOXA5 is up-regulated in U937 cells compared with HL60 cells (RT-PCR assay). (B) Schematic representation of the HOXA5 locus and amplicons used in the ChIP assay. Ex, exon. (C) ChIP analysis of H3K79 dimethylation in the selected HOXA5 regions shown in (B). Input = 1%.
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