Supplementary Figure 1
Loss of one Myb allele has no effect on frequency and reconstitution of B-cell
subsets. (A) Western blot analysis of Myb expression in total bone marrow and
CD19+ cells from Mybf/f and Mybf/d mice; (B) Frequency of pro-B
(B220+CD43+IgM-), pre-B (B220+CD43-IgM-), immature (B220lowCD43-IgM+) and
mature B cells (B220hi CD43-IgM+) in the bone marrow of Mybf/f and Mybf/d mice
(6 mice/each);(C) Contribution of donor marrow cells (CD45.2+) from Mybf/f and
Mybf/d mice to bone marrow and spleen cells of lethally-irradiated CD45.1+
recipient mice; Frequency of pro-B (B220+CD43+IgM-), pre-B (B220+CD43lo/-IgM), immature B (B220loCD43-IgM+) and mature B (B220hiCD43-IgM+) cell subsets
in bone marrow (D) and spleen (E) of lethally-irradiated CD45.1+ recipient mice 8
weeks post-injection of donor bone marrow cells.
Supplementary Figure 2
Representative flow cytometry analysis showing frequency of donor marrowderived B cell subsets found in the bone marrow and spleen of recipient mice.
Cell surface antigens used in identification of B cell subsets are described in
Supplementary Figure 1.
Supplementary Figure 3
Ectopic expression of c-Myb rescues pre-B colony formation of BCR/ABL+Mybw/d
bone marrow cells. (A) pre-B cells (B220+CD43-IgM-) from bone marrow cells
transduced with MigRI/GFP Δ(358-452) c-Myb were sorted for GFP expression
and 45 ng of total RNA was used in real time Q-PCR for detection of Δ(358-452)
c-Myb transcripts. Gapdh expression was determined as control of RNA input.
(B) GFP-positive cells were plated at 100,000 cells/plate in B-cell specific
methylcellulose (n=4 for each). Plates were scored after 10 days. Secondary
plates were seeded at 30,000 cells/plate (n=3) and counted after 10 days (* pvalue <0.05; ** p-value <0.005).
Supplementary Figure 4
Loss of one Myb allele alters global gene expression of p190BCR/ABLtransformed B cells (A) Volcano plot showing gene expression levels between
BCR/ABL+Mybw/f and BCR/ABL+Mybw/d pre-B cells. Red dots represent genes
meeting the criteria of statistical significance (p0.05) and fold-change ([2]), as
indicated by the green lines. Bmi1 is represented as a green dot. (B) Heat map
showing a small portion of the hundreds of genes exhibiting significant change in
expression between the two genotypes. Fold-change was calculated for each
replicate of one group versus the mean expression level of the other group.
Increase in expression is indicated in red, while decrease is indicated in green.
Bmi1 is indicated with an asterisk.
Supplementary Figure 5
Bmi1 is a transcriptional target of c-Myb. Expression of c-Myb enhances
Luciferase activity driven by wild-type (panel A, left) or a MYC binding site
mutated (panel A, right) BMI1 5’ flanking region (nucleotides -233 to +45) but not
by the BMI1 flanking region with a mutated MYB binding site (nucleotides +3 to
+8; CAACTA to CGATTA) (panel B, right). Histograms represent mean + SD of
three (top row) or two (bottom row) experiments performed in triplicate. Basal
activity of plasmid BMI1 (-233 +45) Luc is probably higher than that of plasmid
BMI1 (-233 +23) Luc because the (-233 + 45) BMI1 segment was cloned in the
PGL3- enhancer vector which contains the SV40 enhancer while the (-233 + 23)
BMI1 segment was cloned in the PGL3- basic vector which lacks enhancer
sequences.
Supplementary Figure 6
ChIP assay showing the interaction of c-Myb with the human Bmi-1 promoter. (A)
Schematic diagram of Bmi-1 promoter, 5’ UTR and coding sequence; (B)
Sequence of the -233 to + 43 Bmi-1 promoter with boxed (in bold) MYB binding
site and underlined (in bold) primers used to amplify segments of the BMI-1
promoter; (C) PCR amplification of a segment of the BMI-1 promoter containing a
MYB binding site (nucleotides +3 to +8) from anti c-Myb ChIP DNA. Primers
used for negative control PCR are underlined (in bold) in the sequence shown in
panel A and amplify a region without putative MYB binding sites.
Supplementary Figure 7
Knockdown of c-Myb inhibits proliferation and colony formation of human Ph1ALL cell lines. Two cell lines, Z-181 and SUP-B15 were transduced with
pLVTSH-MYB, a Tet-On MYB shRNA-expression vector, and sorted for the GFP
marker. (A) Western blot analysis showing c-MYB and BMI1 expression in Z-181
and SUP-B15 cells after doxycycline treatment. Cells were lysed 72h postdoxycycline (5g/mL) treatment. (B & C) Proliferation of Z-181 (A) and SUP-B15
(B) cells after induction of MYB knockdown with doxycycline. (C & D) Colony
formation in Z-181 and SUP-B15 cells after induction of MYB knockdown with
doxycycline.
Supplementary Figure 8
Western blot showing Myb and Bmi1 expression levels in pro-B and pre-B cells
of P190BCR/ABL+Mybw/f mice and P190BCR/ABL-Mybw/f littermate controls.