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Supplementary Figure Legends
Supplementary Figure 1. p15 expression and promoter methylation in myeloid
leukemia cell lines. (a) TTGE analysis of p15 promoter methylation. Differentially
methylated sequences within the p15 amplicons were resolved according to differences in
melting temperature and compared to unmethylated (0 of 30 CpG sites methylated) and
methylated (30 of 30 CpG sites methylated) controls, denoted UNMETH + and METH +,
respectively. Agarose gel indicates equal loading of p15 amplicons for TTGE analysis.
(dH2O) was a negative control with no PCR template. (b) TTGE analysis of p15
demethylation at varying doses of 5-Aza-dC (0 – 8 M). Amplicon contains 30 CpG sites
and differentially methylated sequences were resolved according to differences in melting
temperature. Previously characterized KG1a (>95% methylated) and normal CD34 cells
(0% methylated) served as controls for densely methylated (bottom arrow) and
unmethylated (top arrow) p15 promoter alleles, respectively. (c) TTGE analysis of p15
demethylation at varying times in response to 5-Aza-dC (4 M) treatment. Differentially
methylated sequences within the p15 amplicons were compared to the unmethylated
(UNMETH+) and methylated (METH +) controls that contain 0/30 and 30/30 methylated
CpG sites within the p15 promoter amplicon, respectively. Right lower panel shows RTPCR analysis of p15 expression. β-Actin expression was used as a cDNA input control.
Supplementary Figure 2. Pyrosequencing analysis of DNA methylation of p15 and Ecadherin promoters in AML cell lines. (a - h) Pyrosequencing analysis of CpGs in p15
and E-cadherin promoter regions of AML cell lines treated with 5Aza-dC (AML-193:
p15 (1 M), E-cadherin (8 M); Kasumi: p15 and E-cadherin (4 M); KG1a: p15 and Ecadherin (4 M) KG1: p15 and E-cadherin (1 M)), Chaetocin (100 nM), or untreated
(Control). (a) AML-193 – p15; (b) KG1a – p15; (c) Kasumi – p15; (d) KG1 – p15; (e)
AML-193 – E-cadherin; (f) KG1a – E-cadherin; (g) Kasumi – E-cadherin; (h) KG1 – Ecadherin. (i – j) Pyrosequencing analysis of CpGs in p15 and E-cadherin promoter
regions of AML cell lines treated with control shRNA or Suv39h1 shRNA. Y-axis
represents the signal intensity in arbitrary units and the X-axis is the dispensation order.
The sequence analyzed is shown above each chromatogram, where Y represents the
location of the cytosine in the CpG. Dispensations corresponding to the potentially
methylated cytosine (C or T after bisulfite treatment) are highlighted grey. Percentages of
cytosine methylation in different positions are given above respective positions.
Percentage mean methylation of CpGs in p15 and E-cadherin promoters are listed with
standard deviation.
Supplementary Figure 3. Effect of chaetocin on p15 and E-cadherin expression. (a)
Real-time PCR analysis of p15 and E-cadherin expression in KG1a and Kasumi cells
treated with chaetocin. p15 and E-cadherin expression were normalized to HPRT
expression and reported relative to untreated cells. Error bars represent standard deviation
from three independent measurements. (**) represents P-value < 0.01 and (*) represents
P-value < 0.05 between chaetocin treated and untreated cells.
Supplementary Figure 4. Bisulfite sequencing of CpG island promoter regions of p15
and E-cadherin. Bisulfite treated DNA from chaetocin (100 nM) treated and untreated
AML-193 cells were PCR amplified using three primer sets (BS1, BS2, and BS3) for the
CpG island promoter regions of (a) p15 and (b) E-cadherin. CpG islands for p15
(chr9:21998658-21999471) and E-cadherin (chr16:67328536-67329845) were identified
using UCSC genome browser (http://genome.ucsc.edu). Bisulfphite sequencing PCR
primers were designed using MethPrimer (Li LC, Dahiya R. (2002) MethPrimer:
designing primers for methylation PCRs. Bioinformatics 18:1427-1431). The PCR
products for p15 and E-cadherin are shown (BS 1-3). The scale represents the distance
from the transcription start site. Closed and opened circles represent methylated and
unmethylated CpG sites, respectively.
Supplementary Figure 5. Effect of Suv39h1 shRNA and chaetocin on proliferation,
viability, and cell cycle in KG1 cells. (a) MTT analysis of the effect of control shRNA,
Suv39h1 shRNA, and chaetocin on cell proliferation. (b) Trypan blue exclusion assay of
the effect of control shRNA, Suv39h1 shRNA, and chaetocin on cell viability. (c)
Propidium iodide analysis of the effect of control shRNA, Suv39h1 shRNA, and
chaetocin (100 nM) on cell cycle. Cell cycle histograms represent the PI staining of DNA
content in different phases of the cell cycle. Percentages of cells in each phase of the cell
cycle are shown on the histogram. Error bars in (a) and (b) represent standard deviation
from three independent experiments and (*) represents P-value < 0.05 and (**) represents
P-value < 0.01 between treated and untreated cells.