Download Normal human CD34+ haematopoietic progenitor cells

Supplementary Figure legends
Supplementary Figure S1. MYCT1 retroviral expression vectors. Structure of
PINCO GFP control2 and MYCT1 retroviral expression vectors used in this study.
All PINCO vectors contain a CMV promoter-driven GFP expression cassette. Fulllength MYCT1 coding sequence was amplified by PCR from IMAGE clone 40126673
with or without an additional C-terminal HA tag using proof-reading Taq polymerase.
Gel-purified MYCT1 cDNA sequences were cloned into unique BamHI and EcoRI
sites in the PINCO vector for retroviral LTR-driven expression and sequence verified
before use. pA = polyadenylation sequence.
Supplementary Figure S2. Validation of functional MYCT1 expression.
Normal human CD34+ hematopoietic progenitor cells were purified from human cord
blood and transduced with the retroviral expression vectors detailed in Supplementary
Figure S1. (A) Verification of transduction efficiency was determined by flow
cytometric detection of the GFP surrogate marker on day 3 of culture. Threshold
GFP+ expression was defined by autofluorescence of mock-transduced cells. Typical
percentages of viable GFP+ transduced cells are shown for each culture. (B)
Retroviral-driven MYCT1 mRNA expression in day 3 MYCT1 and GFP control
transduced human CD34+ hematopoietic progenitor cell samples was validated by
qRT-PCR, as in Figure 1, using MYCT1-specific primers (Supplementary Material
and Methods). Relative transcript levels were calculated as 1000 x 2-Ct, normalised
to the ABL housekeeping gene.5 (C) Retroviral-driven MYCT1-HA protein
expression in 100g day 3 GFP, MYCT1 and MYCT1-HA transduced CD34+ cell
lysates was validated by Western blotting using an anti-HA antibody and compared
with expression of GAPDH used as a loading control. A representative blot is shown,
demonstrating specific MYCT1-HA reactivity at 22 and 27KDa, corresponding to
alternative translation start sites. (D) Reduced viability of MYCT1 and MYCT1-HA
transduced murine myeloid 32D cells over 48 hours in the absence of growth factor
support. Cells were plated at a density of 1x105 cells/ml in the presence or absence of
WEHI-3B-conditioned medium containing IL-3. At defined time points over 48
hours, cell counts and viability were assessed by trypan blue dye exclusion.
Transduction efficiency was assayed by flow cytometry. Viability at each time point
was normalised to the time zero value for each specific transgene and expressed as a
percentage. MYCT1 and MYCT1-HA cells demonstrated a reduction in viability
compared with GFP control cells, respectively. Data represents mean±plus 1 S.D.
(n=3). Statistical significance was calculated by paired t-test, * P<0.05 and † P<0.01.
Supplementary Figure S3. Reduced viability of MYCT1-expressing monocytes
in low doses of key hematopoietic cytokines. MYCT1 and GFP control transduced
day 4 hematopoietic progenitor cells were subjected to survival assays in the presence
of 10-fold serial dilutions of hematopoietic cytokines in the absence of serum. The
proportion of viable cells (%) was calculated as the number of viable monocytes or
granulocytes recovered after 48 hours/equivalent input populations at time zero. The
proportion of viable cells (%) in each cytokine dose was normalised to the proportion
of viable cells in the absence of cytokine (untreated cells) after 48 hours.
Statistically-significant reductions in the viability of MYCT1-expressing monocytes
compared with GFP control cells were measured in 0.18ng/ml IL-3, 1.32ng/ml SCF,
0.018ng/ml IL-6 and 0.18ng/ml G-CSF. Data represents mean plus±1 S.D. (n=3) and
statistical significance was calculated by paired t-test,*P<0.05 and † P<0.01.
Supplementary Figure S4. MYCT1 over-expression results in increased
frequency and size of monocyte cultures in myeloid-promoting growth
conditions. Colony assays using day 3 GFP control and MYCT1-expressing CD34+
cells were performed by limiting dilution with colonies being scored and analysed for
lineage differentiation after 7 days. (A) The frequencies of monocyte, granulocyte
and mixed granulocyte/monocyte colonies are expressed as percentages of the total
colony numbers. (B) Colony size was normalised to the recovery of APC-conjugated
beads loaded into colony-containing wells before harvest. Data represents mean±plus
1 S.D. (n=3). Statistical significance was not achieved.
Supplementary Figure S5. Expression of the GFI1B blood lineage regulating
transcription factor is significantly down-regulated by RUNX1-ETO and
MYCT1 in hematopoietic progenitor cells. (A) Affymetrix microarray
determination of GFI1B expression (probe-set 208501_at) was performed for pairmatched RUNX1-ETO and GFP control transduced day 3 CD34+ cell samples as in
Figure 1. GFI1B expression is down-regulated in RUNX1-ETO-expressing cells
compared with GFP controls. (B) Quantitative RT-PCR analysis of GFI1B
expression in RUNX1-ETO, MYCT1 and GFP control transduced day 3 CD34+ cells
was conducted as in Figure 1. Expression of GFI1B is down-regulated in RUNX1ETO- and MYCT1-expressing cells compared with GFP controls. (C) GFI1B protein
expression is reduced in MYCT1-expressing day 3 CD34+ cells, as determined by
Western blot. Relative signal intensity (recorded above panels) was generated by
normalising to GAPDH housekeeping signal. (D) MAS5.0-normalised Affymetrix
microarray data demonstrating a down-regulation of GFI1B expression in t(8;21)
acute myeloid leukemia (AML) patients compared with normal donors (Normal) and
with other FAB-M2 AML patients without the t(8;21) translocation (M2). Target
intensity values were subjected to MAS5.0 normalisation, detection call filtering and
a 1.5-fold expression change filter prior to statistical analysis by one-sided ANOVA
using Benjamini-Hochberg correction, ǂ P<0.001. Horizontal bars represent median
values.