SUPPLEMENTARY INFORMATION
In vitro generation of HPCs
Myeloid progenitors derived from methylcellulose cultures of leukemic bone marrow
cells isolated from primary recipients (n=3 for wild-type and 5 for Bmi-1-/- leukemic
mice) were plucked on day 6 and expended in vitro in liquid culture media containing
DMEM (Invitrogen), 15% fetal calf serum (pre-selected, Invitrogen), 6 ng/ml Interleukin
(IL)-3, 10 ng/ml IL-6, 100 ng/ml Steel factor, 100 ng/ml IL-11 (all generated and titrated
at IRCM from COS cell supernatants) and 1x10-5 M β-Mercaptoethanol. Clones that
could generate more than 1010 progeny cells are referred to as highly proliferative clones
(HPCs).
Supplementary Figure 1. Bmi-1 is expressed in CD34+ leukemic cells. a, Cellular
hierarchy in human (xenogenic) acute myeloid leukemia (AML) and phenotypic
presentation of the leukemic hemopoietic stem cells (L-HSCs). b, Bmi-1 is expressed in
purified human CD34++ leukemic cells isolated from AML patients. Proportion of
CD34++ cells represented 35% (AML1), 8% (AML2) and 83% (AML3) of total leukemic
bone marrow cells. Bmi-1 signal intensity was not significantly different between CD34++
cells vs unsorted cells. Twenty thousand cells were isolated from each leukemia sample
(>98% purity for CD34 upon reanalysis), and their total RNA was reverse-transcribed
and PCR-amplified (see methods). Human HL60 cells were used as a positive control for
Bmi-1 expression. Membranes were hybridized to probes specific for Bmi-1
–
Actin and exposed for 48 hrs on a PhosphoImager screen. L-CFC, leukemic colonyforming cells; SP, side population; NO RT, no reverse transcriptase.
Supplementary Figure 2. Bmi-1 regulates the proliferative capacity of stem and
progenitor cells, whether normal or leukemic. This also indicates that Bmi-1 is
dispensable for the genesis of FL (E14.5) HSCs and for their leukemic transformation.
HSC, hemopoietic stem cell; L-HSC, leukemic hemopoietic stem cell; L-Blasts, leukemic
blasts; FL, fetal liver.
Supplementary Figure 3. Expression of selected genes in Bmi-1+/+ (CTRL, n=2) and
Bmi-1-/- Hoxa9-Meis1 highly proliferative clones overexpressing (n=2) or not (n=2) Bmi1. Cell lines were also included as specificity control for hybridization. Total RNA
isolated from each sample was reverse-transcribed and PCR-amplified (see methods).
Membranes were hybridized to probes specific for SCL, MLL, LMO2, Hoxa10, Hoxb4,
Hoxa9, Hoxa5, –Actin and GATA-2 (not shown) and exposed for 36-48 hrs on a
PhosphoImager screen. Note that GATA-2 expression was undetectable in HPCs.
Supplementary Table 1: Generation of gene-specific probes.
Fig.
Probes
Enzymes
Fragments
Bank Number
Accession; Nucleic acids
1, S2
hHOXB4
SalI/PmlI
408 bp
178
AF307160; 1512-1920
3, 5, S2 mHoxa9
PstI/HindIII
243 bp
321
AB005457; 1267-1510
S2
hHOXA10
ApaI
247 bp
84
AF040714; 603-850
S2
hHOXA5
SacII/HindIII
230 bp
185
as described25
S1,S2
hBMI-1
EcoRV/EcoRI
310 bp
508
L13689; 1938-2248
S2
hLMO2
BamH1
476 bp
1587
X61118; 1037-1513
S2
mSCL
PstI/HindIII
437 bp
1590
U01530; 6272-6709
S2
hGATA-2
EcoRI
252 bp
1589
M68891; 1463-1715
S2
hMLL
XbaI/SmaI
936 bp
1586
L04284; 10974-11910
S1,S2
β-Actin
PstI
1.8 kb
212
as described7
1, 6
EGFP
EcoRI/HindIII
719 bp
719
U76561; 289-1008
6
mBmi-1
SacI
1.3 kb
675
as described9