2 Supplementary Figures and Methods
Figure S1: a) Event free survival of AML patients from University Hospital Essen with regard to Gfi1 expression.
b) Gene set enrichment analysis (GSEA) of GFI1-low expressing human leukemic cells with resemblance
to gene expression signature in HSCs NSE=2.2, p=0.0001.
100
100
High GFI1, n= 5
Months
EFS (%) AML Essen
0
Medium GFI1 , n=21
p=0.038 Low GFI1, n=4
500
50
HSC-signature enriched in human low GFI1-expressing AML
a b
Figure S2: Influence of GFI1 level on hematopoietic development. a) Schematic representation of the murine Gfi1 locus and the targeted alleles
b) Western blot of nuclear extracts of Gfi1-WT, Gfi1-36S-KI and Gfi1-KD/KD thymocytes.
a
Mac
1
Gr1
WT KD KI 29 31 71
Granulo- cytes
Mono- cytes
35
WT KI KD
α-Lamin
α-Gfi1 62 49
62
kD
hGFI1 cDNA neo
hGFI1 cDNA
WT
GFI1-KD
GFI1-KI
1.2 2 3 4 5 ATG
41 3
Mac
1
Gr1
WT KD KI 29 31 71
Granulo- cytes
Mono- cytes
35
WT KI KD
α-Lamin
α-Gfi1 62 49
62
kD
hGFI1 cDNA neo
hGFI1 cDNA
WT
GFI1-KD
GFI1-KI
1.2 2 3 4 5 ATG
41 3
Mac
1
Gr1
WT KD KI 29 31 71
Granulo- cytes
Mono- cytes
35
WT KI KD
α-Lamin
α-Gfi1 62 49
62
kD
hGFI1 cDNA neo
hGFI1 cDNA
WT
GFI1-KD
GFI1-KI
1.2 2 3 4 5 ATG
41 3
c d
e
b
f
g h
GFI1-cDNA
GFI1-cDNA
ATC
2
c) Cell surface staining of the BM of the indicated mouse cohorts for Mac1 and Gr1.
d) Wright-Giemsa staining of bone marrow cytospins (bar represents 20 µm).
e) Left: percentage of monocytes in bone marrow (N= 4 Gfi1-WT, N=4 GFI1-KI, N=4 GFI1-KD, N=4) ***
p=0.0002. right: total number of monocytes in bone marrow (N= 4 Gfi1-WT, N=4 GFI1-KI, N=4 GFI1-KD);
*** p=0.0003
f) Left: percentage of granulocytes in bone marrow (N= 4 Gfi1-WT, N=4 GFI1-KI, N=4 GFI1-KD) ****
p<0.0001. Right: total number of granulocytes in bone marrow (N= 4 Gfi1-WT, N=4 Gfi1-KI, N=4 Gfi1-
KD); **** p<0.0001
g) Left: percentage of GMPs (Lin-, Kit+, Sca-, CD34+, CD16/32+ bone marrow cells) (N= 6 Gfi1-WT, N=6
GFI1-KI, N=6 GFI1-KD) *** p=0.007. Right: total number of GMPs (Lin -, Kit+, Sca-, CD34+, CD16/32+ bone
marrow cells) (N= 6 Gfi1-WT, N=6 GFI1-KI, N=6 GFI1-KD) * p=0.04.
h) Left: total number of colonies an in semisolid medium (1000 cells seeded) counted after 7 days. One
representative experiment out of four experiments is shown. Right: total number of cells in semisolid
medium (1000 cells seeded) counted after 7 days. One representative experiment out of four experiments
is shown. **** p<0.0001
3
Figure S3:
a) Schematic representation
of isolating lineage negative cells from
the indicated genotypes and
subsequent transduction with a retrovirus encoding MLL-
AF9 and GFP.
h
4
b) About 1000 GFP-positive cells were seeded in semisolid medium. After 10 days, the number
of colonies was determined in three independent experiments with triplicates for each
experiment. Shown are the results from one representative experiment out of three experiments
(*p=0.025).
c) Similar to B, number of cells in the semisolid medium was determined 10 days after seeding
(*p=0.035).
d) About 3000 GFP-positive cells were seeded in liquid culture. After 7-10 days, number of cells
was determined in three independent experiments with triplicates for each experiment. Shown
are the results from one representative experiment out of three experiments (*p=0.015).
e) WBCs of GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice in comparison to
healthy mice
f) PLTs of GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice in comparison to healthy
mice (**p<0.0045)
g) HGB of GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice in comparison to
healthy mice (*p=0.018)
h) Western Blot of the nuclear extracts of MLL-AF9 leukemic BM cells from GFI1-KI or GFI1-KD
mice. Upper panel subjected to a GFI1 antibody. Lower panel subjected to a lamin antibody.
i) Percentage of blast in the BM of mice transplanted with GFI1-KD or GFI1-KI leukemic cells
(***p=0.0004)
j) Percentage of blast-cells in the blood of mice transplanted with GFI1-KD or GFI1-KI leukemic
cells (*p=0.05). k) Percentage of cKit+ cells in the BM of mice transplanted with GFI1-KD or GFI1-KI leukemic
cells
l) Number of spleen cells from GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice
compared to control mice (*p<0.01)
m) Percentage of Blasts in spleen GFI1-KI-MLL-AF9 and GFI1-KD-MLL-AF9 transplanted mice
5
Bone marrow of MLL-AF9 transplanted mice
' &/ϭ</ͬ </
' &/ϭ<ͬ <
Figure S4: Bone marrow cytospins of transplanted mice with GFI1KI/KI-MLL-AF9 and GFI1KD/KD-MLL-
AF9
6
Figure S5:
a
gfe
b
h
i j k
l m
0
20
40
60
Dead before
300 days
*
Dead after 300 days
Mean fluorescence intensityn
7
a) Western Blot Analysis of nuclear extracts from BM cells of GFI1-KI (WT/KI) and GFI1-KD
(KD/KD) NUP98-HOXD13 transgene mice. The upper panel subjected to a Gfi1 antibody and
the lower panel to a lamin antibody
b) White blood cell count of peripheral blood of GFI1WT/WTxNUP98/HOXD13,
GFI1KD/KDxNUP98/HOXD13 mice and healthy control mice
c) Platelet number peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1KD/KDxNUP98/HOXD13
mice and healthy control mice (*p<0.04); ***p<0.0001)
d) HGB of peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1KD/KDxNUP98/HOXD13 mice and
healthy control mice (**p00.0013; ***p<0.0001)
e) Number of spleen cells from GFI1WT/WTxNUP98/HOXD13, GFI1KD/KDxNUP98/HOXD13 mice in
comparison to healthy mice (*p=0.017)
f) Percentage of Blasts in spleen GFI1-KI/WTxNUP98/HOXD13 and
GFI1-KD/KDxNUP98/HOXD13 (**p=0.0085)
g) RT-PCR detecting Gfi1 mRNA expression levels in bone marrow cells from Gfi1-WT/WT
mice or Gfi1-EGFP/WT heterozygous mice
h) Gfi1 protein expression levels in thymocytes from Gfi1 WT/WT mice or Gfi1 KO/WT
heterozygous mice
i) White blood cell count of peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1-
EGFPxNUP98/HOXD13mice and healthy control mice
i) Platelet number peripheral blood of GFI1WT/WTxNUP98/HOXD13,
GFI1-EGFPxNUP98/HOXD13 mice and healthy control mice (***p≤0.0001)
k) HGB of peripheral blood of GFI1WT/WTxNUP98/HOXD13, GFI1-EGFPxNUP98/HOXD13 mice
and healthy control mice (***p<0.0001)
l) Number of spleen cells from GFI1WT/WTxNUP98/HOXD13, GFI1-EGFPxNUP98/HOXD13 mice
in comparison to healthy mice (*p=0.0096)
m) Percentage of Blasts in spleen GFI1-WT/WTxNUP98/HOXD13 and
GFI1-EGFPxNUP98/HOXD13
n) Mean fluorescence intensity of GFP in blasts of Gfi1-GFP/WT leukemic according to time of
emergence of leukemia.
8
Figure S6:
ba
9
a) GO Biological functions overrepresented amongst genes showing increased acetylation in
Gfi1-KD leukemic samples compared to Gfi1-KI samples, as determined using the DAVID
software. Log p value was used to rank the enrichment.
b) KEGG pathways overrepresented amongst Gfi1 target genes showing increased acetylation
and gene expression in GFI1-KD leukemic samples compared to GFI1-KI samples, identified
using DAVID software. Log p value was used to rank the enrichment
c) Scatter plot comparing the log2 fold change in H3K9ac ChIP-seq between Nup98-HoxD13
Gfi1-KD and Gfi1 KI bone marrow AML samples versus change in mRNA levels between
Nup98-HoxD13 Gfi1-KD and Gfi1 KI bone marrow AML samples.
d) GO Biological functions amongst genes showing increased acetylation and mRNA
expression in Gfi1-KD leukemic samples compared to Gfi1-KI samples, as determined using
DAVID software. Log p value was used to rank the enrichment.
e) KEGG pathways overrepresented amongst GFI1 target genes showing increased acetylation
in GFI1-KD leukemic samples compared to GFi1-KI samples, identified using the DAVID
software. Log p value was used to rank the enrichment.
10
Figure S7: Model of pathophysiological function of low GFI1 expression in AML and as a possible therapeutic intervention. Low level of GFI1 cannot recruit sufficiently histone-modifying enzymes to their target genes. This leads
to the increase acetylation of H3K9 of GFI1 target genes involved in leukemia initiation, leading to
expansion of GMPs. Therapeutic intervention with HDACi could potentially worsen the situation. However,
use of Histone-acetylase inhibitors could rebalance the dysregulated epigenetic modifications
Histone H3GFI1KD
HDAC inhibitorHDAC
HAT
HAT inhibitor
Gfi1 target genes
AMLCMP
Accelerated AML development
Delayed AML formation?OncogenesHistone
acetylation
Ac Ac
GMP
11
Figure S8: New epigenetic therapeutic approach for low GFI1/Gfi1 expressing leukemic cells .
a b c
d
e f
h
d
g
12
a) Lineage negative cells from GFI1-KI or GFI-KD cells were transduced with MLL-AF9 and treated with
the indicated doses of Vorinostat (HDACi) or CTK7a (HATi). One representative experiment out of two
experiments is shown.
b) Treatment of the leukemic cells from the indicated mouse strains with Vorinostat (**p<0.00171;
***p<0.0003; ****p<0.00009). One representative experiment out of two experiments is shown.
c) Treatment of the leukemic cells from the indicated mouse strains with CTK7a (*p=0.015; ***p<0.0006).
One representative experiment out of two experiments is shown.
d) Relative GFI1 m-RNA expression level in Kasumi1 and K562 cells. RNA of Kasumi1 and K562 was
isolated and transcribed into cDNA. The relative expression level of GFI1 m-RNA was analyzed via
quantitative real-time PCR.
e) Kasumi 1 (a myeloid cell line with high Gfi1 expression) and K562 (a myeloid cell line with low Gfi1
expression) were treated with different doses of Vorinostat (*p=0.04; ***p=0.0002). One representative
experiment out of three experiments is shown.
f) Kasumi 1 (a myeloid cell line with high Gfi1 expression) and K562 (a myeloid cell line with low Gfi1
expression) were treated with different doses of CTK7a. (**p=0.0025; ***p=0.0007) One representative
experiment out of three experiments is shown.
g) GFI1 expression in AML-cell lines. *p=0.0186 (N=3 for low GFI1-expressing and n=4 for GFI1-high
expressing cell lines 1
h) Fold change of patient samples used for inhibitor treatment. Fold change relative to Gapdh and
normalized to GFI1 loe expressing patient
i) Scheme of isolating leukemic bone marrow cells from the indicated mouse strains and treated with the
indicated doses of Vorinostat (HDACi) or CTK7a (HATi). One representative experiment out of three
experiments is shown. n=3 for each treatment condition.
j) Treatment of the leukemic cells from the indicated mouse strains with Vorinostat (***p<0.00011;
****p=0.000062). n=3 for each time point. n=3 for each treatment condition
k) Treatment of the leukemic cells from the indicated mouse strains with CTK7a (**p<0.0046;
***p<0.00024****p<0.000031). n=3 for each treatment condition
13
Figure S9: Lineage negative cells from GFI1-KI or GFI-KD cells were treated with CTK7a (HATi) in the
indicated doses. After 48 hours the cell number was determined. Cells are compared to the
control of each genotype.
14
Suppl. Table 1:Patient cohorts from Essen
% all 13 (n=4) 70 (n=21) 17 (n=5) 87 (n=26) - - -Mean Age 60.5 ± 4.2 56.2 ± 2.9 47.2 ± 9.2 54.5 ± 2.9 0.54 0.27 0.44
Gender (% male) 50 (n=2) 48 (n=10) 60 (n=3) 50 (n=13) 0.94 0.77 1.0Cytogenetic low risk (%) 0 (n=0) 19 (n=4) 40 (n=2) 23 (n=6) 0.35 0.19 0.29
Cytogenetic intermediate risk (%) 100 (n=4) 67 (n=14) 60 (n=3) 65 (n=17) 0.19 0.19 0.17Cytogenetic high risk (%) 0 (n=0) 5 (n=1) 0 (n=0) 4 (n=1) 0.65 n.p. 0.69
NA 0 (n=0) 10 (n=2) 0 (n=0) 8 (n=2) 0.52 n.p. 0.56FAB_M0 0 (n=0) 0 (n=0) 0 (n=0) 0 (n=0) n.p. n.p. n.p.
FAB_M1 25 (n=1) 20 (n=4) 40 (n=2) 23 (n=6) 0.82 0.65 0.93FAB_M2 25 (n=1) 14 (n=3) 20 (n=1) 15 (n=4) 0.59 0.86 0.62
FAB_M4 0 (n=0) 28 (n=6) 20 (n=1) 27 (n=7) 0.24 0.37 0.25FAB_M4E 0 (n=0) 5 (n=1) 0 (n=0) 4 (n=1) 0.65 n.p. 0.69
FAB_M5 50 (n=2) 14 (n=3) 20 (n=1) 15 (n=4) 0.11 0.37 0.11
FAB_M6 0 (n=0) 0 (n=0) 0 (n=0) 0 (n=0) n.p. n.p. n.p.
NA 0 (n=0) 20 (n=4) 0 (n=0) 15 (n=4) 0.34 n.p. 0.41
p value between low
and medium+highGFI1 low GFI1 medium GFI1 high
p value between low and medium
p value between low and
highGFI1 medium
+ high
GFI1 low includes patients who express GFI1 lower than 5%. GFI1 medium includes all patients from 6%
to 60% and GFI1 high includes all patients with 61% to 100% GFI1 expression.
Cytogenetic low risk Cytogenetic low risk: inv(16), t(8;21), t(15;17), poor risk: complex aberrations (≥4
anomalies), chromosome 3q, 5, 7 anomalies, inv(3), t(6;11), t(10;11), t(11q23), t(9;22) Intermediate risk:
all other aberrations 2. Presented is the standard error of mean. Student’s t- test is used for significance
of values. For significance of differences between percentages, two sample t- test were used. n.p= not
possible
15
Suppl. Table 2:
AML Patients Cohort from Verhaak et al.
% all 11 (n=32) 82 (n=233) 7 (n=20) 89 (n=253) - - -Mean Age 41.5 ± 2.2 43.24 ± 0.8 38.8 ± 2.3 42.9 ± 0.77 0.43 0.46 0.55
Gender (% male) 47 (n=15) 56 (n=130) 65 (n=13) 57 (n=143) 0.21 0.34 0.28
Cytogenetic low risk (%) 8 (n=2) 19 (n=36) 65 (n=11) 22 (n=47) 0.0006 0.36 0.066
Cytogenetic intermediate risk (%) 44 (n=11) 64 (n=125) 29 (n=5) 61 (n=130) 0.5 0.045 0.066Cytogenetic high risk (%) 48 (n=12) 16 (n=31) 6 (n=1) 15 (n=32) 0.0001 <0.0001 <0.0001
KRAS_mutation 3 (n=1) 1 (n=3) 0 (n=0) 1 (n=3) 0.44 0.34 0.33
IDH1_mutation 3 (n=1) 10 (n=23) 5 (n=1) 10 (n=24) 0.71 0.2 0.2
IDH2_mutation 9 (n=3) 9 (n=21) 5 (n=1) 9 (n=22) 0.6 1.0 1.0
FLT3_TKD_mutation 0 (n=0) 14 (n=32) 5 (n=1) 13 (n=33) 0.21 0.025 0.03
NRAS_mutation 22 (n=7) 9 (n=20) 10 (n=2) 9 (n=22) 0.27 0.026 0.024
FLT3_ITD_mutation 3 (n=1) 33 (n=76) 15 (n=3) 31 (n=79) 0.12 0.0006 0.001
CEBPA_mutation 22 (n=7) 9 (n=22) 0 (n=0) 4 (n=22) 0.029 0.026 0.0001
NPM1_mutation 6 (n=2) 37 (n=87) 15 (n=3) 36 (n=90) 0.26 0.0006 0.0001
EVI1_expression 22 (n=7) 4 (n=9) 0 (n=0) 4 (n=9) 0.029 0.0001 0.0001
FAB_M0 19 (n=6) 2 (n=5) 0 (n=0) 2 (n=5) 0.43 0.0097 <0.0001
FAB_M1 34 (n=11) 22 (n=52) 20 (n=4) 22 (n=56) 0.28 0.13 0.13
FAB_M2 3 (n=1) 22 (n=52) 55 (n=11) 25 (n=63) 0.0001 0.012 0.005
FAB_M4 9 (n=3) 22 (n=51) 10 (n=2) 21 (n=53) 0.9 0.089 0.11
FAB_M4E 6 (n=2) 0 (n=0) 0 (n=0) 0 (n=0) 0.27 0.0002 0.0001
FAB_M5 19 (n=6) 22 (n=52) 15 (n=3) 22 (n=55) 0.71 0.7 0.7
FAB_M6 0 (n=0) 1 (n=2) 0 (n=0) 0 (n=0) n.p. 0.57 n.p.
RAEB 0 (n=0) 2 (n=4) 0 (n=0) 0 (n=0) n.p. 0.42 n.p.
RAEB-T 0 (n=0) 4 (n=10) 0 (n=0) 0 (n=0) n.p. 0.25 n.p.
NA 10 (n=3) 3 (n=7) 11 (n=2) 5 (n=9) 0.91 0.055 0.25
p value (between low and medium +
high)
p value (between low and medium)GFI1 low GFI1 high
p value (between low and
high)GFI1 medium
GFI1 medium +
high
GFI1 low includes patients who express GFI1 lower than 5%. GFI1 medium includes all patients from 6%
to 60% and GFI1 high includes all patients with 61% to 100% GFI1 expression.
Cytogenetic low risk: inv(16), t(8;21), t(15;17), poor risk: complex aberrations (≥4 anomalies),
chromosome 3q, 5, 7 anomalies, inv(3), t(6;11), t(10;11), t(11q23), t(9;22) Intermediate risk: all other
aberrations 2. Presented is the standard error of mean. Student’s t- test is used for significance of values.
For significance of differences between percentages, two sample t- test were used. n.p= not possible
16
Suppl. Table 3: Cox-regressional analysis on prognostic factors
p-Value
Hazard
Ratio
Lower 95%
Confidence
Interval
Higher95%
Confidence
Interval
Caryotype (Good prognosis
caryotype)
,17 0,67 0,4 1,2
NRAS_mutation (present) ,9 1,03 0,6 1,7
NPM1_mutation (present) ,02 0,6 0,4 0,9
EVI1_expression (present) ,09 1,7 0,9 3,2
CEBPA_mutation (present) ,04 0,5 0,27 0,98
Gfi1 expression (low
expression)
,05 3,1 1,01 9,2
Cox regressional multivariate analysis was performed on the cohort presented by Verhaak et al.
(see Supplementary Table 2) including low GFI1 expression.
17
Suppl. Table 4: Patient characteristics of examined samplesName Genotype Gfi1 origin Description
Gfi1-WT WT/WT murine normal WT Gfi1
GFI1-KI KI/KI human murine Gfi1 is replaced by human GFI1
GFI1-KD KD/KD human
murine Gfi1 is replaced by human Gfi1, only 5-15%
expression
Gfi1-EGFP-KI WT/GFP murine one Gfi1 allele is replaced by GFP
Overview about the used mice strains
The table gives an overview about the different mice strains, which were used in these studies. It is
depicted where the Gfi1 is originated from (murine or human). Furthermore a short description about the
exact genotype is given.
For References please see Material and Methods.
18
Suppl. Table 5: Characteristics of patients treated with Vorinostat and CTK7a
Patient Age Sex Genotype Cytogenetic Mutations SourceGfi1 level
1 39 M Gfi1 36S high normal karyotype none bone marrow
2 22 F Gfi1 36S low normal karyotype none bone marrow
F=female, M= male, high= high GFI1 expression, low= low GFI1 expression
For detailed experiment information please see Material and Methods. The Gfi1 mRNA expression was 7-
fold higher in patient 1.
19
Supplementary Material and Methods
Statistical methods
Statistical analysis was done with Graph-Pad Prism 4 or 6 software (La Jolla, CA, USA)
or SPSS version 19 (IBM, Düsseldorf, Germany). The survival rate was calculated using
the Kaplan-Meier method. P values below 0.05 were considered statistically significant.
Differences in percentage were determined using the two-sample t-test. Other statistical
analyses were done using either paired or unpaired two-sided Student´s t-test. The EFS
was assessed unadjusted by stratifying only for expression levels of GFI1. The Cox
proportional-hazards regression modeling was used for age, sex and cytogenetic
findings. For survival of the human cohorts the Log-rank and for murine cohorts the
Mantel Cox test was used. All p values reported are two-sided and considered
significant at 0.05. Due to the exploratory nature of our work, no adjustments were
made for multiple hypothesis testing.
Isolation and culture of hematopoietic progenitors
Lineage-negative (Lin-) cells were isolated from 8-12 week old mice using a Lineage
Cell Depletion Kit (130-090-858, Miltenyi Biotec, Bergisch Gladbach, Germany). For in
vitro studies, Lin- cells were cultured at a density of 1*106 cells/ml in SCM medium
(IMDM (GIBCO) 20% FBS (PAN Biotech GmbH), 1% penicillin-streptomycin (Sigma-
Aldrich, Taufkirchen, Germany) and 20 ng/ml SCF (Miltenyi Biotec Biotec, Cologne,
Germany), 10 ng/ml Interleukin-3 (Miltenyi) and 10 ng/ml Interleukin-6 (Miltenyi Biotec)).
Lin- cells for transplantation were retrovirally transduced using Marrow Max Bone
20
Marrow Medium (GIBCO, Darmstadt, Germany) (20 ng/ml SCF, 10 ng/ml Interleukin-3
and 10 ng/ml Interleukin-6.
Retrovirus production, transduction and transplantation of murine hematopoietic
progenitors
Retroviral transduction of Lin- cells with MLL-AF9 onco-fusion protein-encoding
vectorswas performed at day 3 and 4 after isolation of the cells. Retroviral supernatants
were collected after transient calcium phosphate transfection of 293Tmyc cells with 2.25
μg pCL-Eco retroviral packaging vector (IMGENEX) and 20 µg retroviral plasmid DNA.
Nontissue-treated 48 well plates were coated with 1mg/ml retronectin (Takara Bio Inc.,
Saint-Germaine en Lye, France) overnight at 4°C and blocked with 2% bovine serum
albumin, fraction V (BSA, Sigma) for 30 minutes at room temperature. The virus
supernatant was centrifuged two times for 90 min onto retronectin-coated plates and
Lin- cells were centrifuged for 10 min at 1200 rpm and 4°C. 2µg/ml of polybrene
reagent, Millipore (TR-1003-G) was added and the cells maintained at 37°C in 5% CO 2.
Transduced cells (1*105 GFP-positive cells) were harvested at day 5 after isolation and
injected i.v. into lethally irradiated mice together with 1.5*105 congenic competitive BM
cells.
Flow cytometry analysis
The following antibodies were used for FACS staining: Ly-6G/Ly-6C(Gr-1) (108407),
CD11b (101227), CD8a (100707), CD4 (100433), TER-119/Erythroid Cells Antibody
(116207), CD45R/B220 (103233), CD117(c-Kit) (135105), all from Biolegend (San
21
Diego, CA, USA). Apoptosis was measured by staining the cells with Annexin V
(556419, BD Bioscience, Heidelberg, Germany). For GMP staining, 3*106 BM cells were
stained with CD16/32 PE-Cy7 (101318), Biotin mouse lineage depletion cocktail, Sca-1
FITC (108105 ), CD34 PE (119308), cKit APC (105811), SAv PerCP (45-4317-82),
(ebioscience), Cells were analyzed using FACScan and LSR-II (Becton-Dickinson).
Cell culture
Kasumi1, HL-60 and 293Tmyc cells were obtained from ATCC (Manassas, VA, USA)
and cultured according to supplier’s instructions.
Western Blot
Gfi1 (2.5D17) (1:1000; G6670); Sigma-Aldrich; Anti-Mouse IgG (Goat) (1:10000;
NEF822001EA; Perkin Elmer), Lamin B (C-20) (1:1000; sc-6216; Santa Cruz) and
donkey anti-goat IgG-HRP (1:13000; sc-2020; Santa Cruz) antibodies were used for
Western Blot.
Inhibitoradministration
Vorinostat (SAHA, cat. nr. SML0061-25MG, Sigma-Aldrich) and CTK7a (382115-10mg,
Millipore) were dissolved in DMSO. Approximately 7*104-1*105 cells of peripheral blood
or BM aspirates of AML patients were seeded in 96 well non-tissue culture plates in
IMDM (20% FBS, 1% Pen/Strep and 10 ng/ml human SCF, 10 ng/ml FLT3-Ligand and
10 ng/ml human TPO (Miltenyi Biotec)).
22
RNA-Sequencing and ChIP-Sequencing:
RNA-sequencing:
Sequencing libraries were prepared from RNA extracts using the Illumina TruSeq
Stranded mRNA Kit according to the manufacturer’s instructions, and sequenced using
the TruSeq PE Clusterkit v3-cBot-HS on an Illumina HiSEq 2000 system. Sequencing
reads were aligned to the mm10 genome using Tophat v2.0.10 3. Reads were
processed with Samtools 4 and then mapped to Ensembl transcripts using HTSeq.
Differential expression was tested using the DESeq R package (R Coding Team).
A genome coverage file was generated and scaled to RPM using Bedtools 5 (GEO
accession no. GSE72671).
ChIP-sequencing:
Sequencing libraries were prepared from immunoprecipitated chromatin samples using
the TruSeq DNA kit from Illumina according to the manufacturer’s instructions and
sequenced using the TruSeq PE Clusterkit v3-cBot-HS on an Illumina HiSeq 2000
system.Reads were aligned to the mouse reference mm10 genome using Bowtie2
v2.10 5. Reads were processed and duplicates were removed using Samtools, and
promoter coverage for heatmaps was generated using the annotatePeaks.pl function of
the Homer Software. The CuffDiff R package was used to quantify changes in
acetylation levels on gene promoters.
23
References
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