Supplementary Materials and Methods
Microarray analysis
Microarray scans were inspected to ensure the quality of individual hybridization spots. Spots that
displayed defects which would lead to miscalculation of the intensity ratio were flagged and excluded
from further analysis. The fluorescence intensity for each fluorophore was set as the median intensity of
the pixels enclosed in the perimeter of a spot, minus the median intensity of the background of that
hybridization spot. Hybridization spots were normalized regionally. A lower threshold was established for
each microarray that would exclude 90% of spots containing saline sodium citrate (SSC) buffer, PCR
buffer or Arabidopsis cDNA. Genes determined to be differentially expressed satisfied two conditions.
First, the normalized intensity ratio of the gene had to have a coefficient of variation (standard
deviation/mean) less than 0.20 over a minimum of three replicates. Second, the normalized intensity ratio
had to be a minimum of three standard deviations (SD) greater or less than 1.0.
Confusion Matrix
Our analysis included only those samples where three or more of the cells showed similar cell fates since
we decided that a stipulation of the assay is that cDNA would only be kept if the three other clonal
siblings all displayed the same biological outcome. True positives (TP) were determined by multiplying
the number of cells per clone by the number of clones in which all four sibling cells grew. True negatives
(TN) were determined by multiplying the number of cells per clone by the number of clones in which all
4 sibling cells failed to grew. False positives (FP) were determined from the number of clones in which
all but one cell failed to grow, divided by four since there was only a one in four chance the cell which
failed to grow was selected for PCR. False negatives (FN) were determined from the number of clones in
which all but one cell grew, divided by four since there was only a one in four chance the cell that grew
would have been selected for PCR. Using the data generated in Figure 2c, 112 clones of four cells were
analyzed giving a potential 417 tests (numbers do not add up to 448 because clones with mixed fate had
only a one in four chance of theoretically being selected for analysis).
Cell Lines
Early passages of the OCI/AML-4 cell lines (obtained from Dr. M.D. Minden, University Health
Network, Toronto, Canada) were cultured on an irradiated OP9 feeder layer in IMDM with thioglycerol, 5% v/v fetal bovine serum (FBS) and supplemented with recombinant human (rh)G-CSF
(50ng/mL), rhIL-11 (30ng/mL), rhFlt3 ligand (30ng/mL), rhGM-CSF (30ng/mL), rhIL-6 (1ng/mL), 3%
v/v conditioned medium of Chinese hamster ovary (CHO) cells transfected with a vector expressing
murine c-kit ligand (KL-CM) (D. Donaldson, Genetics Institute, Cambridge, MA), insulin (10g/mL),
transferrin (5g/mL), and 0.5% v/v bovine serum albumin (BSA). Cultures were passaged twice weekly
to maintain a density of 2 x 105 cells/mL. To limit selection for highly proliferative subclones through
continuous passaging, cultures were discarded after approximately three months of passage. U937 cells
were purchased from ATCC and grown in RPMI supplemented with 10% FBS. 32Dcl3 cells were
purchased from ATCC and grown in RPMI with 1ng/mL of rmIL-3. The 293GPG retroviral packaging
cell line (a gift of Richard Mulligan, Harvard University) was grown in DMEM medium supplemented
with 10% FBS, tetracycline (1 mg/mL), G418 (0.3mg/mL) and puromycin (2 mg/mL).
Growth factor drop out experiments
Growth factor requirements of the OCI/AML-4 cell line was established at either 14 days (Figure 1a) or
23 days (Figure 1B) by assessing proliferative ability following growth factor drop-out. All experimental
groups were cultured without a feeder layer in Iscove’s modified Dulbecco’s medium plus 5ug/mL
transferrin, 0.5% bovine serum albumin, 10ug/mL insulin, monothioglycerol and 5% fetal bovine serum.
Positive controls (the first bar in the graph) were additionally supplemented with recombinant human
(rh)G-CSF, murine c-kit ligand, rhIL-7, rhIL-11, rhFlt-3 ligand, rhIL-6 or rhGM-CSF. From this starting
point growth factors were individually deleted. Cells were seeded at a density of 30 cells per well in 100
μL of appropriate medium and scored for growth after either 14 days (A) or 23 days (B). Viable
collections containing a minimum of 120 cells were scored positive. Each experimental group contained
48 replicates.
Limiting Dilution Analysis
The relative number of clonogenic cells in the OCI/AML-4 cell populations was inferred by culturing
cells at limiting dilutions and scoring colony growth. Cells were seeded at limiting dilutions in liquid
cultures on top of a feeder layer, OP-9 and each well was monitored for growth weekly under a light
microscope. The frequency of cells able to continuously proliferate for 3 and 6 weeks respectively was
determined from the percentage of wells negative for growth at those respective time points using Poisson
statistics{Fazekas de St, 1982 #68}.
Real-time PCR
The following primer sequences were used:
Human NR2F6:
Fwd: 5’-TCTCCCAGCTGTTCTTCATGC-3’
Revs: 5’-CCAGTTGAAGGTACTCCCCG-3’
Human GAPDH:
Fwd: 5’-GGCCTCCAAGGAGTAAGACC -3’
Revs: 5’-AGGGGTCTACATGGCAACTG-3’.
3’ end Mus NR2F6:
Fwd: 5’-CCTGGCAGACCTTCA ACAG -3’
Revs: 5’-GATCCTCCTGGCCCATAGT -3’
3’ end Mus L32:
Fwd: 5’-GCCATCAGAGTCACCAATCC-3’
Revs: 5’-AAACATGCACACAAGCCATC -3’
shRNA hairpins
Sense shRNA hairpin sequences were as follows:
mus shNR2F6.1
5’- GAT CCG CAT TAC GGC GTG TTC ACC TTC AAG AGA GGT GAA CAC GCC GTA
ATG CTT TTT TCT AGA G 3’
mus shNR2F6.2
5’ –GAT CCG CAA CCG TGA CTG TCA GAT TAA GTT CTC TAA TCT GAC AGT CAC
GGT TGT TTT TTC TAG AG-3’
mus shNR2F6.3
5’- GAT CCG TGT CCG AGC TGA TTG CGC ATT CAA GAG ATG CGC AAT CAG CTC
GGA CAT TTT TTC TAG AG-3’
human shNR2F6.1
5’-GAT CCG CAT TAC GGT GTC TTC ACC TTC AAG AGA GGT GAA GAC ACC GTA
ATG CTT TTT TCT AGA G-3’
human shNR2F6.2
5’-GAT CCG CCT CTG GAC ACG TAA CCT ATT CAA GAG ATA GGT TAC GTG TCC
AGA GGT TTT TTC TAG AG- 3’