Published Ahead of Print on February 2, 2017, as doi:10.3324/haematol.2016.156257.
Copyright 2017 Ferrata Storti Foundation.
Loss of FOXM1 promotes erythropoiesis through increased
proliferation of erythroid progenitors.
by Minyoung Youn, Nan Wang, Corinne LaVasseur, Elena Bibikova, Sharon Kam,
Bertil Glader, Kathleen M. Sakamoto, and Anupama Narla
Haematologica 2017 [Epub ahead of print]
Citation: Youn M, Wang N, LaVasseur C, Bibikova E, Kam S, Glader B, Sakamoto KM, and
Narla A. Loss of FOXM1 promotes erythropoiesis through increased proliferation of erythroid progenitors.
Haematologica. 2017; 102:xxx
doi:10.3324/haematol.2016.156257
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Loss of FOXM1 promotes erythropoiesis through increased proliferation of erythroid
progenitors
Minyoung Youn1, Nan Wang1, Corinne LaVasseur1, Elena Bibikova1, Sharon Kam1, Bertil
Glader1, Kathleen M. Sakamoto1* and Anupama Narla1*
1
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA
*
Co-senior corresponding authors
Running head: FOXM1 in Erythropoiesis
Corresponding author: Anupama Narla, Department of Pediatrics, Stanford University School
of Medicine, Stanford, CA 94305
e-mail: [email protected]
Tel: 650-723-5535
Fax: 650-723-5231
Word count: abstract 184; main text 3,767; figures 6; supplemental files 1.
Acknowledgments
The authors would like to thank Narla Mohandas for critical discussion, reading of our
manuscript, and sharing the RNA-seq data. This research was funded by NIH R01HL097561,
R01DK107286, Department of Defense BM110060 (K.M.S.), K08 DK090145-06 (A.N), the
Child Health Research Institute at Stanford Postdoctoral Fellowship 1111239-280-JHACT
(M.Y.), Stanford Bio-X Undergraduate Summer Research Program (S.K.), and USHHS Ruth
L. Kirschstein Institutional National Research Service Award # T32 CA009056 (E.B.).
Abstract
FOXM1 belongs to the fork head/winged-helix family of transcription factors and
regulates a network of proliferation-associated genes. Its abnormal upregulation has been
shown to be a key driver of cancer progression and an initiating factor in oncogenesis.
FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation,
suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact
molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still
uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord
blood CD34+ cells were transduced with FOXM1 shRNA lentivirus. Knockdown of FOXM1
resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally,
knockdown of FOXM1 increased BrdU incorporation in erythroid cells, suggesting greater
proliferation of erythroid progenitors. We also observed that the defective phosphorylation of
FOXM1 by CHK2 or CDK1/2 increased the erythroid population in a manner similar to
knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, FDI-6, increased red
blood cell number through increased proliferation of erythroid precursors. Overall, our data
suggest a novel function of FOXM1 in normal human hematopoiesis.
Introduction
Hematopoiesis is the critical process by which normal blood cells are derived from
self-renewing and pluripotent hematopoietic stem cells (HSCs)1, 2. Erythropoiesis, which
leads to the formation of mature red blood cells, requires that each cell division is
simultaneously coupled with differentiation3, 4. Erythropoiesis can be divided into 3 stages:
early erythropoiesis, terminal erythroid differentiation, and reticulocyte maturation5, 6. During
early erythropoiesis, HSCs sequentially give rise to a common myeloid progenitor,
megakaryocyte-erythrocyte progenitor, burst-forming unit-erythroid (BFU-E), and colonyforming unit-erythroid (CFU-E) cells that differentiate into proerythroblasts7-11. In terminal
erythroid differentiation, morphologically recognizable proerythroblasts undergo sequential
mitosis to become basophilic, polychromatic, and orthochromatic erythroblasts that expel
their nuclei to become reticulocytes12. At the final step of erythropoiesis, multinuclear
reticulocytes mature into red blood cells accompanied by loss of intracellular organelles,
decrease in cell volume, and extensive membrane remodeling13-18.
Erythropoiesis is tightly regulated by various regulatory growth factors and
transcription factors3, 6, with erythropoietin (EPO) and GATA-1 playing essential roles
7, 10
.
The binding of EPO to its receptor (EPOR) activates Jak2-Stat5 signaling pathway to
promote the proliferation of erythroid progenitor cells and to rescue erythroid progenitors
from cell death
19-21
. GATA-1 leads to the expression of erythroid-specific genes including
EPOR, adult globin genes, heme biosynthesis enzymes, and erythroid membrane proteins22.
The expression or transcriptional activation of erythroid-specific growth and transcription
factors is critical for normal erythropoiesis.
FOXM1 belongs to the fork head/winged-helix family of transcription factors and
binds to a specific DNA consensus sequence through a highly conserved DNA-binding
domain (DBD)23-26. It is a key transcription factor in the regulation of a network of
proliferation-associated genes including the G1/S transition, S progression, G2/M transition,
and M progression, and is also critical for DNA replication, mitosis, spindle assembly, and
genomic stability24,
27-30
. Consistent with its role in cell-cycle progression, FOXM1
expression is highly upregulated in a number of human cancers including liver, ovarian,
breast, prostate, colon, and brain tumors31, 32. Its abnormal upregulation has been shown to be
a key driver of cancer progression and an initiating factor of oncogenesis33.
FOXM1 is also highly expressed in multipotent progenitor cells and inhibits
differentiation of progenitors, suggesting that FOXM1 plays a role in the maintenance of
multipotent progenitor cells 34-36. It is reported that Foxm1 participates in the maintenance of
pluripotency of mouse P19 embryonal carcinoma cells by directly regulating Oct4
transcription
37
. In addition, the overexpression of Foxm1 alone in human newborn
fibroblasts restarts the expression of pluripotent genes, including Oct4, Nanog, and Sox2.
Recently, it was also reported that Foxm1 is essential for the quiescence and maintenance of
hematopoietic stem cells in the mouse model
38
. Loss of Foxm1 induced the decrease of
cyclin-dependent kinase inhibitors by suppressing Nurr1 gene (a critical regulator of HSC
quiescence) expression. However, the exact molecular mechanisms by which FOXM1
regulates human hematopoietic stem and progenitor cells are still uncharacterized.
In this study, we have examined the role of FOXM1 in normal hematopoiesis using
human cord blood CD34+ cells and lentivirus to target FOXM1. We found that knockdown of
FOXM1 resulted in an increase in the erythroid population compared to the myeloid
population, with higher expression of CD71+ (erythroid) cells compared to CD11b+ (myeloid)
cells. We also found that FOXM1 knockdown increased BrdU incorporation in only CD71+
cells, suggesting greater proliferation of erythroid progenitors. Taken together, these studies
suggest a novel function of FOXM1 in normal human hematopoiesis. Our data indicate that
FOXM1 inhibitors, such as FDI-6, may be beneficial in treating patients with anemia due to
decreased red blood production.
Methods
Cell culture
Primary human CD34+ hematopoietic stem/progenitor cells were purified from umbilical
cord blood units obtained from The National Cord Blood Program at the Howard P. Milstein
Cord Blood Center of New York Blood Center. These are unprocessed, non-clinical grade
cord blood units and are considered “Research Units”. These materials are de-identified and
are therefore considered not to involve human subjects as per the Stanford Human Research
Protection Program and Institutional Review Board. CD34+cells were purified using MACS
cell separation (Miltenyi Biotec) and cryopreserved. Primary human bone marrow CD34+
cells were purchased from Lonza. Upon thawing, cells were cultured in x-Vivo15 medium
(Lonza) containing 10% FBS, 1x Penicillin-Streptomycin-Glutamine (PSG) (Invitrogen),
FLT-3 (Miltenyi Biotec), TPO (Miltenyi Biotec), IL-3 (Miltenyi Biotec), IL-6 (Miltenyi
Biotec), SCF (Miltenyi Biotec), EPO, and transferrin (Sigma-Aldrich) by 3 phase liquid
culture system as described in Supplemental Table S1. HEK 293 and K562 cell lines were
cultured in Iscove's Modification of Dulbecco's Medium (Corning) containing 10% FBS and
1x PSG.
Colony assays
GFP+ or mCherry+ sorted hematopoietic cells were seeded in methylcellulose medium
containing IL-3, SCF, GM-CSF, and EPO (H4434, StemCell Technologies), in triplicate, with
a density of 1000 cells per plate. Erythroid (BFU-E and CFU-E) and myeloid (CFU-G/M)
colonies were counted 2 weeks later by an investigator blinded to the conditions.
Methylcellulose medium containing only EPO (H4330, StemCell Technologies) was used for
erythroid colony (CFU-E) and colonies were counted 1 week later. Methylcellulose medium
containing SCF, IL-3, G-CSF, and GM-CSF (H4035, StemCell Technologies) was used for
myeloid colony and colonies were counted 2 weeks later.
Flow cytometry
For cell surface flow cytometry, cells were washed with PBS and then incubated with
indicated antibodies for 20 minutes at room temperature. After washing, cells were analyzed
by FACS. For intracellular flow cytometry, cells were fixed in 3.2% paraformaldehyde for 10
minutes at 37°C, and permeabilized with 100% methanol for 30 minutes at -80°C. After
washing, cells were incubated with indicated antibodies and then analyzed by FACS. Data
were collected on a FACS Calibur (BD Biosciences) or a DxP10 (Cytek) flow cytometer
and analyzed using FlowJo Software (v.10). All antibodies are listed in Supplemental
Methods.
BrdU incorporation
Cells were plated in a 48 well plate and incubated with 10 μl of 1 mM BrdU (BD Pharmingen)
for 45 minutes. Cells were fixed in 3.2% paraformaldehyde for 10 minutes at 37°C, and
permeabilized with 100% methanol for 30 minutes at -80°C. After washing, cells were treated
with RQ1 RNase-free DNase (Promega) for 1 hour at 37°C. After washing, cells were
incubated with BrdU and CD71-PE antibodies and then analyzed by FACS. Antibody against
BrdU (Bu20A: APC-conjugated: 17-5071-42) was purchased from eBioscience.
Drug treatment
FDI-6 (Axon2384; Axon Medchem) was dissolved in dimethylsulfoxide (DMSO) to create a
40 mM stock and added to cells at final concentration of 10 μM. Roscovitine (S1153;
Selleckchem), U0126-EtOH (S1102; Selleckchem), and ADZ7762 (S1532; Selleckchem)
were dissolved in DMSO and added to cells at final concentration of 10 μM, 5 μM, and 50
nM, respectively. Cells were treated with the respective drugs from day 1 to day 5 after
thawing; the drug was then washed out for the remainder of the culture time.
Results
FOXM1 is highly expressed in the erythroid population
To understand the role of FOXM1 in hematopoietic cells, we first investigated the
expression profiles of FOXM1 in individual cell populations. Trilineage differentiation of
human cord blood CD34+ hematopoietic progenitor cells was induced in vitro using a 3phase liquid culture system, as described in Supplemental Table S1. We used the following
cell surface markers: CD71 for early erythroid, glycophorin A (GlyA) for late erythroid,
CD33 for early myeloid, CD11b for late myeloid, and CD41a for megakaryocytes.
Populations were analyzed on day 6 or day 12 of culture. Sorting efficiencies for each
population were confirmed by running RT-qPCR with each cell marker (Supplemental Figure
S1). We found that FOXM1 had a 3-fold increased RNA level in CD71+ cells compared to
CD11b+ cells (Figure 1A). To observe the effects on the protein level, we performed
intracellular flow cytometry for FOXM1 with each cell surface marker. Consistent with the
RNA levels, FOXM1 protein was high in the CD71+ population, suggesting that FOXM1
plays a specific role in erythropoiesis (Figure 1B and Supplemental Figure S2).
Data from a previously published paper confirms our findings that FOXM1 has a
role in erythropoiesis. The authors performed a detailed transcriptome analysis of the various
stages of normal human erythropoiesis including BFU-E, CFU-E, proerythroblasts (Pro),
Early and Late basophilic erythroblasts (E Baso and L Baso), polychromatic erythroblasts
(Poly), and orthochromatic erythroblasts (Ortho)
39
. Interestingly, FOXM1 expression was
elevated in erythroid cells, suggesting a functional role for FOXM1 in erythroid population
(Figure 1C; data courtesy of An X. et al). We chose to focus on the earlier stages of
erythropoiesis which also demonstrated higher expression of FOXM1 since it was
challenging to study the later stages of erythropoiesis in our liquid culture system using
shRNA knockdown.
FOXM1 downregulation increases the erythroid population
To understand the function of FOXM1 in human erythropoiesis, we examined
whether FOXM1 knockdown affects normal hematopoiesis. We confirmed the knockdown
efficiency of two different shRNAs against FOXM1 and observed significantly decreased
RNA levels in human cord blood CD34+ cells (Figure 2A). We also confirmed decreased
protein level with FOXM1 knockdown in the K562 cell line by immunoblot analysis and in
the human cord blood CD34+ cells by intracellular flow cytometry (Supplemental Figure
S3A and B). After transduction of human CD34+ cells with lentivirus expressing FOXM1
shRNA, we studied hematopoietic differentiation using FACS analysis with a range of cell
surface markers. We found that knockdown of FOXM1 resulted in an increase of the
erythroid population as measured by CD71 and GlyA and a decrease in the myeloid
population as measured by CD11b (Figure 2B, Supplemental Figure S4, and Supplemental
Figure S5). Additionally, we detected effects on specific stages of erythroid differentiation by
using GlyA, CD49d, and Band3 as markers
40
. Consistent with our findings in the
CD71+/GlyA+ population, the populations of Pro, E Baso, and L Baso stages were increased
by FOXM1 knockdown (Supplemental Figure S6A and B). Similarly, methylcellulose colony
assays demonstrated increased numbers of CFU-E colonies and decreased numbers of CFUGM colonies but we did not note any morphological changes in FOXM1 knockdown cells
(Figure 2C/2D and Supplemental Figure S7).
To determine whether the increase in the erythroid population resulting from
FOXM1 knockdown was not due to a decrease in myelopoiesis, we cultured human cord
blood CD34+ cells in medium supporting either erythroid or myeloid differentiation as
described in Supplemental Table S2 and S3. After transduction with lentivirus expressing
FOXM1 shRNA, we measured the colony forming activity of cells cultured in
methylcellulose media specific for either erythroid progenitors or myeloid progenitors. We
observed an increase in CFU-E colonies with erythroid specific medium in CD34+ FOXM1
knockdown cells but no change in CFU-GM colonies with myeloid media (Figure 2E). We
observed similar results using FACS analysis (Supplemental Figure S8). These results
suggest that FOXM1 knockdown directly affects erythropoiesis.
To study the effects of FOXM1 overexpression during erythropoiesis, we cloned
full-length cDNA of FOXM1 into a lentiviral vector (Supplemental Figure S9A and B).
Human CD34+ cells were transduced with lentivirus expressing FOXM1 cDNA and FACS
analysis and colony assays were performed. FOXM1 overexpression did not affect erythroid
or myeloid differentiation (Supplemental Figure S9C and D).
Taken together, these results suggest that FOXM1 knockdown directly increases the
erythroid population and overexpression does not increase erythroid or myeloid colony
formation.
FOXM1 downregulation increases the proliferation of erythroid progenitors
FOXM1 is known to play a critical role in cell proliferation 37. In order to understand
its potential mechanism of action in erythropoiesis, we investigated its role on the
proliferation of hematopoietic progenitor cells. We again used an in vitro liquid culture
system to differentiate and separate transduced CD34+ cells into erythroid and non-erythroid
or myeloid populations using CD71 and CD11b. Viable cells were counted every 3 days to
observe effects on proliferation. We found that FOXM1 knockdown increased the number of
CD71+ cells, but not CD71-, CD11b+, or total cell populations after 11 days in culture
(Figure 3A and B). This is similar to the increase in the CD71+/GlyA+ population, which we
noted in earlier experiments (Figure 2B). To verify that the increased cell number is caused
by increased cell proliferation, we performed BrdU incorporation assays at 13 days after
transduction and found that knockdown of FOXM1 resulted in increased BrdU positive cells
in the CD71+ population only, indicating more rapid proliferation of erythroid progenitors
(Figure 3C left panel and Supplemental Figure S10). Similarly, H3P-ser10, which is a
specific marker for M phase, was increased in FOXM1 knockdown CD71+ cells (Figure 3C
right panel), indicating increased mitotic activity in the erythroid population with knockdown
of FOXM1. Additionally, we transduced sorted CD71+ erythroid cells with FOXM1 shRNA
lentivirus and observed increased BrdU positive cells (Supplemental Figure S11), confirming
the direct action of FOXM1 knockdown in erythroid proliferation.
In summary, these data suggests that FOXM1 downregulation increases the
proliferation of the erythroid progenitors.
FOXM1 effects on erythropoiesis are independent of the p53 pathway
Previous reports established that p53 and FOXM1 negatively regulate each other’s
activity 27, 41. In our system, we confirmed that FOXM1 expression was decreased in CD34+
progenitor cells treated with Nutlin-3, a drug that leads to p53 stabilization by blocking
MDM2 (Supplemental Figure S12A and B). It has also been reported that FOXM1-deficient
MEFs have increased transcriptional activity of p53 with corresponding stimulation of their
target gene expression
27
. This suggests the possibility that p53 activity is involved in
regulating FOXM1 activity during erythropoiesis. To test this hypothesis, we studied the
expression of p53 target genes in FOXM1 knockdown cells. FOXM1 downregulation did not
affect the expression of p21, WIG-1, BAX, and GADD45A in either the early or late stages of
erythropoiesis (Supplemental Figure S13A). The expression of GATA1 (a canonical erythroid
transcription factor) and TNF-α (whose role in erythropoiesis we and others have studied)
were also not affected (Supplemental Figure S13B)
42
. Finally, we could not detect any
increase in the p53 protein level and cPARP+ apoptotic cells in FOXM1 knockdown cells,
although their levels were increased in RPS19 knockdown cells, which were used as a
positive control (Supplemental Figure S13C and D) 42, 43.
Taken together, these results suggest that the effects of FOXM1 on erythropoiesis are
p53-independent.
FOXM1 function on erythroid proliferation requires its phosphorylation by CHK2 or
CDK1/2 kinases
It is known that FOXM1 activity is regulated by phosphorylation involving multiple
regulatory kinases. For example, FOXM1 transcriptional activity requires binding of the
CDK-Cyclin complexes and subsequent phosphorylation to regulate G2/M cell cycle
regulatory genes
44-46
. Additionally, FOXM1 is phosphorylated by DNA damage-induced
CHK2, resulting in stabilization of the FOXM1 protein 27.
To understand the effects of the upstream signaling pathways on FOXM1 during
erythroid proliferation and differentiation, we studied three representative kinases, checkpoint
kinase 2 (CHK2), cyclin-dependent kinase 1/2 (CDK1/2), and polo-like kinase 1 (PLK1). We
examined their expression levels at various time points during hematopoiesis using our in
vitro CD34+ system. All observed kinases were expressed at the protein level throughout
hematopoiesis, with a relatively high expression in the earlier stage (Figure 4A). We then
made alanine-substituted FOXM1 mutants on serine or threonine residues that would be
predicted to affect normal phosphorylation (Figure 4B). We confirmed that mRNA and
protein of the FOXM1 mutants were normally expressed in CD34+ cells by RT-qPCR and in
HEK 293 cells by immunoblot analysis (Supplemental Figure S14A and B). We then
transduced CD34+ progenitor cells with these constructs and performed methylcellulose
colony assays as described previously. We found increased BFU-E and CFU-E colonies in
mt1, mt2/3, and mt1-5 but not mt4/5 (Figure 4C), suggesting that FOXM1 phosphorylation
by CHK2 and CDK1/2 kinases is important in its role in regulating the proliferation of
erythroid progenitors. Similarly, we observed increased BFU-E at 5 days, increased CFU-E
with decreased BFU-E at 8 days, and increased Pro at 11 days after culture with all mutant
forms, indicating an effect on promoting erythropoiesis (Supplemental Figure S15). Finally,
we studied whether these kinase inhibitors show similar effects to FOXM1 mutants. We
treated CD34+ cells with Roscovitine (CDK1/2 inhibitor) or ADZ7762 (CHK1/2 inhibitor)
and we then performed FACS analysis on the indicated days. We observed an increase in the
CD71+/GlyA+ population and a decrease in the CD11b+ population upon treatment with the
kinase inhibitors (Figure 4D and Supplemental Figure S16). Additionally, we found similar
results with U0126-EtOH, an inhibitor of MEK1/2, which is the upstream kinase of CDK1/2.
These results indicate that CHK2 or CDK1/2 signaling pathways are required for
FOXM1 function on erythroid differentiation.
FDI-6 increases the erythroid population through increased proliferation of its progenitors.
Recently, Gormally et al identified a small molecule, FDI-6, as a specific inhibitor of
FOXM1
47
. FDI-6 inhibits the transcriptional activity of FOXM1 by its displacement from
target gene promoters. To observe whether FDI-6 shows same effects to FOXM1
downregulation on erythroid cells, we treated cord blood CD34+ cells with 10 μM of FDI-6
for 5 days. We confirmed that 10 μM of FDI-6 was enough to inhibit FOXM1 transcriptional
activity in cord blood CD34+ cells by observing the expression of several known FOXM1
target genes, FOXM1, CDC25B, CyclinD1, CENPF, CyclinB2, and p27 (Supplemental Figure
S17). We performed FACS analysis every 3 days and found dramatic increases of CD71+ and
CD71+/GlyA+ populations with FDI-6 treatment (Figure 5A and Supplemental Figure S18).
Similar results were observed in methylcellulose colony assays containing 10 μM of FDI-6
with increased numbers of CFU-E colonies and decreased numbers of CFU-GM colonies
with treatment (Figure 5B). Finally, we studied the effects of FDI-6 on cellular proliferation
as described in Figure 3 and noted that FDI-6 increased the number of CD71+ cells and BrdU
positive CD71+ cells (Figure 5C, D, and Supplemental Figure S19).
These results suggest that inhibition of FOXM1 by FDI-6 increases erythroid cells
by enhancing erythroid progenitor proliferation.
In summary, inhibition of FOXM1 activity either through decreased phosphorylation,
expression (knockdown) or transcriptional activity (chemical inhibitor), increased
proliferation of erythroid progenitors, thereby promoting erythropoiesis (Figure 6).
Discussion
In this study, we found that decreased levels of the FOXM1 protein increased red
blood cell production through increased proliferation of human erythroid progenitors. We
found that knockdown of FOXM1 by shRNA resulted in an increased number of erythroid
cells with decreased myeloid cells. FOXM1 expression was relatively low in the myeloid
population, and we suspect that the reduction in the myeloid population is an indirect effect
from the promotion of erythropoiesis in our in vitro system. Indeed, we observed that the
myeloid population was not affected by FOXM1 downregulation in isolation (Figure 2E, 3B,
and Supplemental Figure S8). In contrast, there is significant expression of FOXM1
throughout normal human erythropoiesis suggesting that FOXM1 plays a critical role and
may be a unique target for certain forms of anemia.
Since FOXM1 is normally associated with proliferation, we would predict that
knockdown would inhibit clonal expansion of stem/progenitor cells inhibiting differentiation.
Interestingly, our results demonstrated that knockdown of FOXM1 increased proliferation of
erythroid progenitors. Our findings suggest that FOXM1 could be a repressor of
erythropoiesis. FOXM1 has been shown to function as a transcriptional repressor to inhibit
mammary luminal differentiation
48
. FOXM1 directly represses the mRNA expression of
GATA3, a master regulator of luminal differentiation, through a RB-dependent and DNMTdependent mechanism. We tested whether FOXM1 also has a role as repressor in our in vitro
model of hematopoiesis. We observed mRNA levels of known FOXM1 target genes such as
proliferation-associated genes including the cell cycle regulatory genes (CyclinD1, p27,
CyclinB1/B2, CyclinA1/A2, PCNA, CDC25b, PLK1, NEK2, CENPF) and the proliferation
genes (TGF-α, JNK1, IGF-1, NEDD4) in FOXM1 knockdown cells. However, we did not
observe any significant changes in their gene expression by FOXM1 knockdown in pure
CD71+ erythroid population as well as in the all mixed CD71+ and CD71- population (data
not shown). We therefore suspect that the effects of FOXM1 on the proliferation of erythroid
progenitors are due to alternative mechanisms.
We studied the effects of FOXM1 on erythroid population using shRNA knockdown,
mutant proteins, and drug inhibitor. There was some discordance among the phenotypes,
specifically with regard to the colony forming assays. While FOXM1 knockdown increases
CFU-E numbers only, FOXM1 mutants exhibit increased BFU-E and CFU-E. Although we
examined three representative kinases, there are still multiple regulatory pathways for
FOXM1 activity such as protein stability by ubiquitin-proteasome pathway and protein
expression by miRNAs. Therefore, it is conceivable that regulation of FOXM1 function in
erythroid progenitors may not solely rely on its phosphorylation.
We also studied the effects of CHK2 and CDK1/2 kinase inhibitors on erythropoiesis.
These kinases are well-known to regulate various pathways, including cell cycle pathways
and we, therefore, expected that it would be difficult to observe the effects of kinase
inhibition on erythropoiesis. For this reason, we initially used a range of drug concentrations
and found that most of the cells were dead or arrested at the higher concentrations. However,
we did observe an increase in the erythroid population and a decrease in the myeloid
population without any cell growth defects at the lower concentrations, which is similar to
our results with the FOXM1 mutants which cannot be phosphorylated by these kinases.
Previous work in zebrafish models using morpholino antisense against foxm1 showed
a blood defect in 70% of embryos, which is not consistent with our results 49. We hypothesize
that FOXM1 may have different functions during embryogenesis in zebrafish or due to
effects in the bone marrow microenvironment. There are heterogeneous cells in the bone
marrow microenvironment, and they regulate each other through paracrine as well as
autocrine mechanisms. Currently, optimal human cell model systems to study the extrinsic
effects of FoxM1 in vitro are not available.
In addition, Hou et al previously reported that in vivo deletion of Foxm1 in the
mouse leads to more proliferation and fewer quiescent cells of CD34+ cells but did not affect
the differentiation of mature blood cells or the frequency of erythroid blasts38. One
consideration is the source of CD34+ cells (i.e cord blood for our studies and bone marrow
for previous studies). We did examine the effects of FOXM1 knockdown and FOXM1
inhibitor, FDI-6 in human bone marrow CD34+ cells and we could still observe the same
effects (Supplemental Figure S20). Therefore, the difference in our findings is likely due to
the variability of FOXM1 expression. The previous paper showed higher expression of
Foxm1 in primitive hematopoietic cells than in differentiated cells including myeloid cells, B
cells, erythroblasts, and T cells. However, we observed that FOXM1 is highly expressed in
more differentiated hematopoietic progenitors, especially erythroid progenitor. Our data
suggest that FOXM1 plays a unique role in erythroid cells rather than CD34+ cells. In
addition, there may be differences between the mouse and human hematopoietic systems 39.
In summary, our results demonstrate a novel function of FOXM1 in normal human
hematopoiesis, in which FOXM1 deficiency leads to increased proliferation of erythroid
progenitors, resulting in increased erythroid differentiation. These results suggest that
FOXM1 inhibitors such as FDI-6 may be beneficial in treating certain patients with anemia.
Disclosures
Contribution: M.Y., N.W., C.L., E.B., and S. K. performed the experiments with
human hematopoietic cells. Research was designed by M.Y., K.M.S., and A.N. The
manuscript was written by M.Y., B.G., K.M.S., and A.N.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
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Figure Legends
Figure 1. FOXM1 is highly expressed in the erythroid population. Human cord blood
CD34+ hematopoietic progenitor cells were cultured in vitro in differentiation media. At the
indicated days, cells were stained with each cell surface marker and sorted. (A) RNA was
collected from the indicated populations and analyzed by RT-qPCR. (B) At the indicated days,
cell were stained with each cell surface marker and FOXM1 antibody. Stained cells were
analyzed by flow cytometry. (C) Data courtesy of An X. et al (please see Ref for
experimental details). Error bars represent standard deviations of 4 independent replicates.
Significance was evaluated using Student t test. *p<0.05, **p<0.01, ***p<0.001
Figure 2. FOXM1 downregulation increases the erythroid population. Human cord blood
CD34+ hematopoietic progenitor cells were transduced with lentivirus carrying shRNA
against FOXM1 or luciferase (Luc) control. (A) Cells were sorted for GFP+ at 5 days after
transduction. RNA was collected and analyzed by RT-qPCR. (B) Transduced cells were
analyzed for CD71, GlyA, and CD11b expression by flow cytometry at the indicated days
after transduction. (C) 1000 cells of GFP+ cells were plated in methylcellulose media and
cultured for 2 weeks. Colonies were counted by an investigator blinded to the conditions. (D)
GFP+ cells followed cytospin and Wright-Giemsa staining at the indicated days posttransduction, and imaged at 63 x magnification. Arrow indicates a representative erythrocyte.
(E) Cells in erythroid media or myeloid media were transduced with lentivirus and then were
sorted for GFP+ at 5 days after transduction. 1000 cells of each GFP+ cells were plated in
methylcellulose media for detection of CFU-E or CFU-GM, and cultured. Colonies were
counted by an investigator blinded to the conditions. *p<0.05, **p<0.01, ***p<0.001
Figure 3. FOXM1 downregulation increases the proliferation of erythroid progenitors.
Human cord blood CD34+ hematopoietic progenitor cells were transduced with lentivirus
carrying shRNA against FOXM1 or Luc control. Cells were sorted for CD71+/GFP+ (for
CD71+ group), CD71-/GFP+ (for CD71- group), CD11b+/GFP+ (for CD11b+ group), or
GFP+ (for all group) cells at 5 days after transduction. (A and B) 4000 sorted cells were
cultured for additional 12 days or 9 days. Cells were counted every 3 days. (C) Sorted cells
were cultured for additional 8 days. At 13 days post-transduction, cells were incorporated
with BrdU, and then stained with BrdU and CD71 antibodies. Or, cells were stained with
H3P-ser10 and CD71 antibodies. Stained cells were analyzed by flow cytometry. *p<0.05,
**p<0.01, ***p<0.001
Figure 4. FOXM1 function on erythroid proliferation requires its phosphorylation by
CHK2 or CDK1/2 kinases. (A) Human cord blood CD34+ hematopoietic progenitor cells
were cultured for 21 days. At the indicated days, cells were harvested. Protein was collected
and analyzed by immunoblot analysis. GATA1 was used as an erythroid-specific marker
protein. β-Actin was used as a loading control. (B) The Schema of human FOXM1
phosphorylation sites by CHK2, CDK1/2, and PLK1. (C) Human cord blood CD34+
hematopoietic progenitor cells were transduced with lentivirus carrying FOXM1 mutant
cDNA. Cells were sorted for mCherry+ at 5 days after transduction. 1000 cells of mCherry+
cells were plated in methylcellulose media and cultured for 2 weeks. Colonies were counted
by an investigator blinded to the conditions. (D) Human cord blood CD34+ hematopoietic
progenitor cells were treated with the each kinase inhibitor for 5 days. Cells were analyzed
for CD71, GlyA, and CD11b expression by flow cytometry at the indicated days after
washing out the drug. *p<0.05, **p<0.01, ***p<0.001
Figure 5. FDI-6 increases the erythroid population through increased proliferation of its
progenitors. Human cord blood CD34+ hematopoietic progenitor cells were treated with
FDI-6 for 5 days. (A) The drug was washed out after 5 days. Cells were analyzed for CD71
and GlyA expression by flow cytometry at the indicated days after culture. (B) 1000 drugtreated cells were plated in methylcellulose media containing 10 μM of FDI-6 and cultured
for 10 days. Colonies were counted by an investigator blinded to the conditions. (C) Cells
were sorted for CD71+ or CD71- cells at 5 days after drug treatment. 2250 sorted cells were
cultured for an additional 9 days in differentiation media. Cells were counted every 3 days.
(D) Sorted cells were cultured for an additional 4 days in differentiation media. At 9 days
post-culture, cells were incorporated with BrdU, and then stained with BrdU and CD71
antibodies. Stained cells were analyzed by flow cytometry. *p<0.05, **p<0.01, ***p<0.001
Figure 6. Model of increased erythroid production by FOXM1 deficiency. FOXM1 is
functionally activated through the CHK2 or CDK1/2 kinase cascade. When FOXM1 cannot
be activated because of certain mutations or defective expression, the proliferation of
erythroid progenitors is amplified, resulting in increased erythroid cell production. We also
observed that the FOXM1 inhibitor, FDI-6, shows a similar effect to FOXM1downregulation.
Supplemental Data
Supplemental Methods
Lentiviral transduction
The shRNA constructs targeting human FOXM1 were cloned into a pLVTH-GFP vector.
Target sequences are listed in Supplemental Table S4. Full-length cDNA encoding human
FOXM1 (isoform 2) was obtained by RT-PCR from HEK 293 cells and cloned into pCHDGFP or pCHD-mCherry vectors. The FOXM1 point mutants were generated from the wildtype mCherry-FOXM1 vector by site-directed mutagenesis.
HEK 293 cells were transfected with lentiviral vectors by using CalPhos Mammalian
Transfection Kit (Clontech) according to the manufacturer's instruction. After 3 days of
transfection, culture media containing lentivirus were collected and concentrated with
ultracentrifugation through a 10% sucrose solution. Viruses were resuspended in 1x Hanks'
Balanced Salt Solution (Corning) overnight at 4°C and kept at -80°C before use.
Primary CD34+ cells were transduced by spinoculation with lentivirus at a multiplicity of
infection score of 10 after 24 hours in culture. Cells were sorted for GFP or mCherry after 5
days of culture using a BD FACSAida (BD Biosciences) and harvested for downstream
assays as indicated in the results.
Flow cytometry
For cell surface flow cytometry, cells were washed with PBS and then incubated with
indicated antibodies for 20 minutes at room temperature. After washing, cells were analyzed
by FACS. Antibodies against CD71-PE (clone MA712;555537), CD71-APC (clone
MA712;551373),
CD71-BV421
(clone
MA712;562995),
CD71-APC-H7
(clone
MA712;563671), GlyA-APC (clone GA-R2;551336), GlyA-PE-Cy7 (clone GA-R2;563666),
CD33-PE-Cy5 (clone WM53;551377), CD11b-PE (clone ICRF44;555388), CD41a-PE (clone
HIP8;555467), CD34-PE (clone581;555822), and CD36-APC (clone CB38;550956) were
purchased from BD Biosciences. Antibodies against CD11b-APC (clone ICRF44;17-0118042)
and CD123-PE-Cy7 (clone 6H6;25-1239-42) were purchased from eBioscience. Antibody
against CD11b-BV605 (clone ICRF44;301332) was purchased from BioLegend. Antibody
against CD49d (Integrin alpha4)-PE (clone MZ18-24A9;130-093-282) was purchased from
Miltenyl Biotec. Antibody against Band3-APC was kindly provided by Dr. Xiuli An at
NYBC.
For intracellular flow cytometry, cells were fixed in 3.2% paraformaldehyde for 10 minutes at
37°C, and permeabilized with 100% methanol for 30 minutes at -80°C. After washing, cells
were incubated with indicated antibodies and then analyzed by FACS. Antibody against
FOXM1 (ab55006) was purchased from Abcam. Antibodies against p53 (1C12:Alexa Fluor
647 Conjugate:2533) and H3P-ser10 (11D8: Alexa Fluor 647 Conjugated:650805) were
purchased from Cell Signaling and BioLegend, respectively.
Data were collected on a FACS Calibur (BD Biosciences) or a DxP10 (Cytek) flow cytometer
and analyzed using FlowJo Software (v.10).
RT-qPCR
Total RNA was extracted by using TRIzol reagent (Invitrogen). 500 ng of total RNA was
transcribed into first strand cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). Real time
qPCR reaction was run with iQTM SYBR Green MasterMix (Bio-Rad) using the CFX384
TouchTM Real-Time PCR Detection System (Bio-Rad). 7SL scRNA was used as an internal
control. Fold change of mRNA was calculated using the ΔΔCt method. All primer sets used
are indicated in Supplemental Table S5.
Immunoblot analysis
Cells were resuspended with RIPA buffer (50 mM Tris-Cl(pH8.0), 150 mM NaCl, 0.1% SDS,
0.5% Deoxycholate, 1% NP-40, 1 mM EDTA, protease inhibitors). Total cell lysates were
resolved in SDS-polyacrylamide gels and transferred to polyvinylidene flouride (PVDF)
membranes. The membranes were blocked with 5% skim milk in TBST, and primary
antibody was bound overnight at 4°C. After washing with TBST, horseradish peroxide
(HRP)-conjugated secondary antibody (Bio-Rad) was bound for 1 hour at room temperature.
Immunoreactive signals were detected with WesternBright ECL HRP substrate (Advansta).
Antibodies against FOXM1 (clone D12D5;5436), GATA1(clone D52H6;3535), CHK2 (clone
D9C6;6334), CDK2 (clone 78B2;2546), MEK1/2 (clone L38C12;4694) and PLK1 (clone
208G4;4513) were purchased from Cell Signaling. Antibody against -Actin (A5316) was
purchased from Sigma-Aldrich and antibody against p21(clone C-19;sc-397) was purchased
from Santa Cruz Biotechnology.
Cytospin and Wright-Giemsa staining
1x105 sorted cells in 500 l were used to prepare cytospin preparations on coated slides,
using the Shandon Southern Cytospin Centrifuge. The slide were stained with Wright-Giemsa
solution for 5 minutes, then stained with 25:1 of Phosphate buffer (pH6.8) colorant Selon
Giemsa (Harleco) for 10 minutes, then stained with 6:1 of Phosphate buffer (pH6.8):WrightGiemsa solution for 1 minute, and finally rinsed in water. The cells were imaged using Leica
DMLB microscope and INSIGHT SPOT2 software.
Statistical analysis
Data are presented as mean ± s.d. P values for statistical significance were obtained using an
unpaired Student t-test. P < 0.05 was considered significant. The data are representative of at
least two independent experiments.
Table S1. 3 Phase Liquid Culture System
Basic media 0 ~5 days
6 ~ 10 days
50ng/ml SCF
50ng/ml SCF
x-Vivo15
50ng/ml TPO
50ng/ml TPO
10% FBS
50ng/ml FLT-3
50ng/ml FLT-3
1x PSG
20ng/ml IL-6
20ng/ml IL-6
20ng/ml IL-3
20ng/ml IL-3
3U/ml EPO
250g/ml Transferrin
Table S2. Erythroid Medium
Basic media 0 ~5 days
50ng/ml SCF
x-Vivo15
20ng/ml IL-3
10% FBS
0.5U/ml EPO
1x PSG
11 days ~
50ng/ml SCF
50ng/ml TPO
50ng/ml FLT-3
20ng/ml IL-6
20ng/ml IL-3
3U/ml EPO
1mg/ml Transferrin
6 ~ 10 days
50ng/ml SCF
20ng/ml IL-3
3U/ml EPO
250g/ml Transferrin
11 days ~
50ng/ml SCF
20ng/ml IL-3
3U/ml EPO
1mg/ml Transferrin
Table S3. Myeloid Medium
Basic media 0 day~
50ng/ml SCF
x-Vivo15
50ng/ml TPO
10% FBS
15ng/ml G-CSF
1x PSG
20ng/ml GM-CSF
Table S4. shRNA Sequences
shRNA
Target Sequence
5'- CGCTGAGTACTTCGAAATGTC-3'
shLuc
5'- GGACCACTTTCCCTACTTTAA-3'
shFOXM1(1)
5'- GCAAGATCCTGCTGGACAT-3'
shFOXM1(2)
5'- CTACGATGAGAACTGGTT CTA-3'
shRPS19(1)
Table S5. Primer Sequences for RT-qPCR
Gene
Forward
7SL scRNA 5'-ATCGGGTGTCCGCACTAAGTT-3'
5'-ATACGTGGATTGAGGACCACT-3'
FOXM1
5'-GCTGCTTTCCCTTTCCTTGC-3'
CD71
5'-TACGCACAAACGGGACACATA-3'
GlyA
5'-GGTGTGACTACGGAGAGAACC-3'
CD33
5'-ACTTGCAGTGAGAACACGTATG-3'
CD11b
5'-GATGAGACCCGAAATGTAGGC-3'
CD41a
5'-ATCCCGTGTTCTCCTTT-3'
p21
5'-AGAAGCCTTTTGGGCAGGAG-3'
WIG-1
5'-TGACATGTTTTCTGACGGCAAC-3'
BAX
Reverse
5'-CAGCACGGGAGTTTTGACCT-3'
5'-TCCAATGTCAAGTAGCGGTTG-3'
5'-CTGCTCGTGCCACTTTGTTC-3'
5'-TCGTTCCAATAACACCAGCCA-3'
5'-GGTAGGGTGGGTGTCATTCC-3'
5'-TCATCCGCCGAAAGTCATGTG-3'
5'-GTCTTTTCTAGGACGTTCCAGTG-3
5'-GCTGGCATGAAGCC-3'
5'-TGCTGCATAGTAATTTCGGAGTT-3'
5'-GGAGGCTTGAGGAGTCTCACC-3'
GADD45A
GATA-1
TNF-
CDC25B
CyclinD1
CyclinB2
CENPF
P27
5'-GAGAGCAGAAGACCGAAAGGA-3'
5'-CCTCATCCGGCCCAAGAAG-3'
5'-CCCAGGGACCTCTCTCTAATC-3'
5'-ACGCACCTATCCCTGTCTC-3'
5'-TATTGCGCTGCTACCGTTGA-3'
5'-CCGACGGTGTCCAGTGATTT-3'
5'-ACCTTCACAACGTGTTAGACAG-3'
5'-AACGTGCGAGTGTCTAACGG-3'
5'-CAGTGATCGTGCGCTGACT-3'
5'-CCATCCTTCCGCATGGTCAG-3'
5'-AGCTGCCCCTCAGCTTGAG-3'
5'-CTGGAAGCGTCTGATGGCAA-3'
5'-CCAATAGCAGCAAACAATGTGAAA-3'
5'-TGTTGTTTTGGTGGGTTGAACT-3'
5'-CTGAGGCTCTCATATTCGGCA-3'
5'-CCCTCTAGGGGTTTGTGATTCT-3'
Figure S1.
Figure S1. Each cell population expresses its specific cell surface marker. Human cord
blood CD34+ hematopoietic progenitor cells were cultured for 6 days or 12 days. At the
indicated days, cells were stained with each cell surface marker and sorted. RNA was
collected from the indicated populations and analyzed by RT-qPCR.
Figure S2.
Figure S2. FOXM1 is highly expressed in erythroid progenitors. Human cord blood
CD34+ cells were cultured in vitro in differentiation media. At day 6, cells were stained with
CD71 or CD33 cell surface marker and sorted. Protein was collected from each population
and analyzed by immunoblot analysis. GATA1 was used as an erythroid-specific marker
protein. -Actin was used as a loading control.
Figure S3.
Figure S3. FOXM1 knockdown decreased FOXM1 protein level. (A) K562 cell line was
transduced with lentivirus carrying shRNA against FOXM1 or Luc control. Protein was
collected and analyzed by immunoblot analysis at 5 days after transduction. -Actin was used
as a loading control. (B) Human cord blood CD34+ cells were transduced with lentivirus
carrying shRNA against FOXM1 or Luc control. At 5 days after transduction, cells were
stained with FOXM1 antibody and were analyzed by flow cytometry. ***P<0.001
Figure S4.
Figure S4. FOXM1 downregulation increases the erythroid population. Human cord
blood CD34+ cells were transduced with lentivirus carrying shRNA against FOXM1 or Luc
control. Cells were analyzed for CD71, GlyA, and CD11b expression by flow cytometry at
the indicated days after transduction. The numbers of cells expressing CD71, GlyA, and
CD11b within about 5000 GFP+ cells are shown. **P<0.01, ***P<0.001
Figure S5.
Figure S5. FOXM1 knockdown increases the CD71+/GlyA+ population and decreases
the CD11b+ cell population. Human cord blood CD34+ cells were transduced with
lentivirus carrying shRNA against FOXM1 or Luc control. Cells were analyzed for CD71,
GlyA, and CD11b expression by flow cytometry at 14 days after transduction. Sample flow
plots are shown.
Figure S6.
Figure S6. FOXM1 downregulation increases specific stages of later erythroid
population. Human cord blood CD34+ cells were transduced with lentivirus carrying shRNA
against FOXM1 or Luc control. Cells were analyzed for GlyA, CD49d, and Band3 expression
by flow cytometry at the indicated days after transduction. (A) Sample flow plots at 11 days
are shown. (B) The percentages of each population are shown. *p<0.05, **p<0.01,
***p<0.001
Figure S7.
Figure S7. FOXM1 downregulation does not affect morphological changes of colonies.
Human cord blood CD34+ cells were transduced with lentivirus carrying shRNA against
FOXM1 or Luc control. Cells were sorted for GFP+ at 5 days after transduction. 1000 cells
of GFP+ cells were plated in methylcellulose media and cultured. Colonies were imaged
using a phase contrast microscope at 5 x magnification.
Figure S8.
Figure S8. FOXM1 has the direct effect on erythroid cells. Human cord blood CD34+
cells were cultured in erythroid medium or myeloid medium. At 1 day after culture, cells
were transduced with lentivirus carrying shRNA against FOXM1 or Luc control and were
cultured for additional days in each media. Transduced cells were analyzed for CD71, GlyA,
and CD11b expression by flow cytometry at the indicated days after transduction. **p<0.01,
***p<0.001
Figure S9.
Figure S9. FOXM1 overexpression does not affect erythroid differentiation. Human cord
blood CD34+ cells were transduced with lentivirus carrying full-length human FOXM1
cDNA or GFP control. Cells were sorted for GFP+ at 5 days after transduction. (A) RNA was
collected and analyzed by RT-qPCR. (B) HEK 293 cell line was transfected with FOXM1
cDNA or GFP control. Protein was collected and analyzed by immunoblot analysis at 5 days
after transduction. -Actin was used as a loading control. (C) Transduced cells were analyzed
for CD71 and GlyA expression by flow cytometry at the indicated days after transduction. (D)
1000 cells of GFP+ cells were plated in methylcellulose media and cultured for 2 weeks.
Colonies were counted by an investigator blinded to the conditions. **p<0.01
Figure S10.
Figure S10. FOXM1 downregulation increases the proliferation of erythroid progenitors.
Human cord blood CD34+ cells were transduced with lentivirus carrying shRNA against
FOXM1 or Luc control. Cells were sorted for CD71+/GFP+ (for CD71+ group), CD71/GFP+ (for CD71- group), or GFP+ (for all group) cells at 5 days after transduction. Sorted
cells were cultured for an additional 8 days. Cells were incorporated with BrdU, and then
stained with BrdU and CD71 antibodies. Stained cells were analyzed by flow cytometry. The
numbers of cells expressing CD71, GlyA, and CD11b within about 18000 GFP+ cells are
shown. **P<0.01, ***P<0.001
Figure S11.
Figure S11. FOXM1 knockdown in erythroid progenitors increases BrdU+ population.
Human cord blood CD34+ cells were cultured for 5 days and then sorted for CD71+. Sorted
cells were transduced with lentivirus carrying shRNA against FOXM1 or Luc control. After
culturing an additional 8 days, cells were incorporated with BrdU, and then stained with
BrdU. Stained cells were analyzed by flow cytometry. ***p<0.001
Figure S12.
Figure S12. p53 negatively regulates FOXM1 expression. Human cord blood CD34+ cells
were cultured for 4 days and then treated with Nutlin-3 (N6287, Sigma-Aldrich) for an
additional 24 hours. (A) RNA was collected and analyzed by RT-qPCR. p21 was used as a
positive control. (B) Protein was collected and analyzed by immunoblot analysis. p21 was
used as a positive control and -Actin was used as a loading control.
Figure S13.
Figure 13. FOXM1 function on erythropoiesis is independent of the p53 pathway.
Human cord blood CD34+ cells were transduced with lentivirus carrying shRNA against
FOXM1 or Luc control. (A and B) Cells were sorted for GFP+ at the indicated days after
transduction. RNA was collected and analyzed by RT-qPCR. (C) Transduced cells were
stained with p53 and CD71 antibodies at the indicated days after transduction. Stained cells
were analyzed by flow cytometry. RPS19 downregulation was used as a positive control. (D)
Sorted cells were cultured for an additional 8 days. At 13 days post-transduction, cells were
stained with cPARP antibody and analyzed by flow cytometry. RPS19 downregulation was
used as a positive control. *p<0.05, **p<0.01
Figure S14.
Figure S14. FOXM1 mutant mRNA and protein were expressed in human
hematopoietic progenitor cells. (A) Human cord blood CD34+ cells were transduced with
lentivirus carrying FOXM1 mutant cDNA. Cells were sorted for mCherry+ at 5 days after
transduction. RNA was collected and analyzed by RT-qPCR. (B) HEK 293 cell line was
transfected with FOXM1 mutant cDNA. Protein was collected and analyzed by immunoblot
analysis at 3 days after transfection. -Actin was used as a loading control.
Figure S15.
Figure S15. FOXM1 mutants increase specific stages of erythroid population. Human
cord blood CD34+ cells were transduced with lentivirus carrying FOXM1 mutant cDNA.
Cells were analyzed for CD123, CD34, and CD36 expression or for GlyA, CD49d, and
Band3 expression by flow cytometry at the indicated days after transduction. The percentages
of each population are shown. *p<0.05, **p<0.01, ***p<0.001
Figure S16.
Figure S16. FOXM1 function on erythroid proliferation requires its phosphorylation by
CHK2 or CDK1/2 kinases. Human cord blood CD34+ cells were treated with the each
kinase inhibitor for 5 days. Cells were analyzed for CD71, GlyA, and CD11b expression by
flow cytometry at the indicated days after washing out the drug. The numbers of cells
expressing CD71, GlyA, and CD11b within about 20000 alive cells are shown. *p<0.05,
**P<0.01, ***P<0.001
Figure S17.
Figure S17. Expression of FOXM1 target genes was decreased by FDI-6 treatment.
Human cord blood CD34+ cells were treated with FDI-6. Cells were collected at 5 days after
treatment. RNA was collected and analyzed by RT-qPCR. *p<0.05, **p<0.01, ***p<0.001
Figure S18.
Figure S18. FDI-6 increases the erythroid population. Human cord blood CD34+ cells
were treated with FDI-6 for 5 days, and then the drug was washed out. Cells were analyzed
for CD71 and GlyA expression by flow cytometry at the indicated days after culture. The
numbers of cells expressing CD71 and GlyA within about 20000 alive cells are shown.
**P<0.01, ***P<0.001
Figure S19.
Figure S19. FDI-6 increases proliferation of erythroid progenitors. Human cord blood
CD34+ cells were treated with FDI-6 for 5 days. Cells were sorted for CD71+ or CD71- cells
at 5 days after drug treatment. Sorted cells were cultured for an additional 4 days in
differentiation media. At 9 days post-culture, cells were incorporated with BrdU, and then
stained with BrdU and CD71 antibodies. Stained cells were analyzed by flow cytometry. The
numbers of BrdU+ cells within about 5000 alive cells are shown. *P<0.05, ***P<0.001
Figure S20.
Figure S20. FOXM1 downregulation increases the erythroid population in human bone
marrow CD34+ hematopoietic progenitor cells. (A) Human bone marrow CD34+ cells
were transduced with lentivirus carrying shRNA against FOXM1 or Luc control. Transduced
cells were analyzed for CD71, GlyA, and CD11b expression by flow cytometry at the
indicated days after transduction. (B) Human bone marrow CD34+ cells were treated with
FDI-6 for 5 days and then the drug was washed out. Cells were analyzed for CD71 and GlyA
expression by flow cytometry at the indicated days after culture. *p<0.05, **p<0.01,
***p<0.001