1
2
Table 1S. Primer information.
3
4
Table 2S. MPN patient information.
5
6
Fig 1S. Volcano plot of miRNA deregulation in MPN patient CD34+ cells. Yellow:
7
miRNAs not expressed in controls but expressed in MPN patients. Red: miRNAs
8
expressed in controls but not expressed in MPN patients. Blue: miRNAs with
9
deregulated expression level.
10
11
Fig 2S. miRNA array validation in MPN patient CD34+ by qRT-PCR. Same RNA
12
samples used the Taqman miRNA array was utilized in single qRT-PCR to confirm the
13
expression levels of miR-22*, miR-133b and miR-9.
14
15
Fig 3S. Heat map represents the Pearson correlation between patient and control
16
samples based on raw Ct values. High correlation coefficient indicates similar miRNA
17
expression profile.
18
19
Table 3S. List of deregulated miRNAs in MPN CD34+ cells with known function in
20
hematopoietic systems.
21
22
Table 4S. Commonly deregulated miRNAs in MPN CD34+ cells and MPN
23
granulocytes (p<0.01). A subset of the deregulated miRNAs are also differentially
24
expressed in an independent miRNA array study using MPN patient granulocytes
25
(n=39, 25 PV, 14 ET).
26
27
Table 5S. 27 miRNAs were significantly deregulated in PV (n=3) compared to
28
controls (n=4) (p<0.01).
29
30
Table 6S. 116 miRNAs were significantly deregulated in ET (n=3) compared to
31
controls (n=4) (p<0.01).
32
33
Fig 4S. Identification of JAK2V617F-independent miRNAs. (A) HEL cells were
34
treated with JAK inhibitor (2uM) (Calbiochem, Rockland, MA) or DMSO. Number of live
35
cells was determined by Trypan Blue at 12 hrs (left panel). miRNA was extracted at 12
36
hrs and quantified by qRT-PCR (right panel). Data are expressed as the mean ± SEM
37
of biological triplicates. (B) HEL cells were treated with JAK inhibitor (2uM)
38
(Calbiochem, Rockland, MA) or DMSO. Number of live cells was determined by Trypan
39
Blue at 24 hrs (left panel). miRNA was extracted at 24 hrs and quantified by qRT-PCR
40
(right panel). Data are expressed as the mean ± SEM of biological triplicates.
41
42
Fig 5S. miRNA responsiveness to manipulation of JAK2 activity in TF1 cells and
43
JAK2V617F knock-in mice. (A) The JAK2 lentiviral construct uses a two-promoter
44
system, with GFP driven by the EF1 promoter and A2 driving JAK2 (gift of Dr. Carl
45
June, University of Pennsylvania). Human wild-type JAK2 and JAK2V617F cDNA was
46
cloned downstream of the A2 promoter using NheI and SalI sites. (B) JAK2WT and
47
JAK2V617F viral particles were produced by transient transfection of 293T cells with
48
EF1-GFP-JAK2WT/V617F, pRSV-REV, pMDLg/RRE and pGMV/VSVG constructs
49
using Fugene6 (Roche). After 24h, medium was replaced with fresh DMEM, and virus
50
was collected at 48h. TF-1 cells were stably transduced with control 2A lentivirus or
51
JAK2V617F. Infected TF-1 cells were cultured in the absence of cytokines for 22 hrs
52
before mRNA was extracted and quantified for JAK2 expression by RT-PCR. (C) Cells
53
were cultured with or without cytokines for 4 days and cell density was determined by
54
MTT assay (right panel). (D) Total RNA was extracted from the stably transduced TF-1
55
cells and miRNA expression levels were determined by RT-PCR. Data are expressed
56
as the mean ± SEM of technical triplicates(*p<0.05). (E) After euthanization, the femurs
57
of JAK2 transgenic mice were flushed with PBS RBCs were removed with lysis buffer
58
(Sigma-Aldrich, St. Louis, MO), and remaining cells were washed and pelleted. Total
59
RNA was extracted using the MirVana kit, and miRNA levels were evaluated by qRT-
60
PCR.
61
62
Fig 6S. miR-433 expression is not changed during myelopoiesis and
63
megakaryopoiesis from BM CD34+ cells. (A) CD34+ cells were cultured in SFEM
64
supplemented with cytokines to promote myeloid (50ng/ml SCF, 10ng/ml IL-3, 10ng/ml
65
IL-6 and 10ng/ml GMCSF) lineages for 12 days. miRNA was extracted on days 3, 6, 9
66
and 12 and quantified by RT-PCR. Data are expressed as the mean ± SEM of
67
biological triplicates. (B) CD34+ cells were cultured in SFEM supplemented with
68
cytokines to promote and megakaryocytic (50ng/ml TPO, 10ng/ml IL-6, 10ng/ml IL-3)
69
lineages for 12 days. miRNA was extracted on days 3, 6, 9 and 12 and quantified by
70
RT-PCR. Data are expressed as the mean ± SEM of biological triplicates.
71
72
Fig 7S. miR-433 expression validation. (A) Validation of miR-433 upregulation in
73
MPN patient BM CD34+ cells by RT-PCR. Un-amplified RNA from patient and control
74
BM CD34+ cells was used for RT-PCR quantification. (B) CD34+ cells were stably
75
transduced with pmiR empty vector, pmiR scrambled control, or pmiR-433 lentivirus,
76
and cultured in erythroid liquid culture for 9 days. miRNA was extracted on day 8 and
77
miR-433 expression level was assessed by RT-PCR. Due to the low cell number, cells
78
from biological triplicates were pooled together for RT-PCR, and data are expressed as
79
the mean ± SEM of technical triplicates. (C) CD34+ cells were stably transduced with
80
pmiR empty vector, pmiR scrambled control, or pmiR-433 lentivirus, and cultured in
81
mythylcellulose supporting erythroid and myeloid colony formation for 14 days. miRNA
82
was extracted on day 14 and miR-433 expression level was assessed by RT-PCR.
83
84
Figure 8S. miR-433 negatively regulates myeloid and CFU-MK colony formation.
85
(A) CD34+ cells were stably transduced with miR-433 or scrambled control lentivirus.
86
4x103 GFP sorted cells were seeded in 3ml of Methocult® H4434 medium (Stem Cell
87
Technologies) and duplicate 1.4ml aliquots were plated into 35-mm dishes (Corning,
88
Corning, NY). Three independent experiments were performed, and colony number
89
was assessed on D14. miR-433 colony formation is presented as a relative ratio to
90
pmiR-scr, and data are expressed as the mean ± SEM of biological triplicates (p<0.05).
91
(B) CD34+ cells were stably transduced with miR-433 or scrambled control lentivirus,
92
and plated in Megacult-C supporting CFU-MK colony formation. Two independent
93
experiments were performed. Slides were fixed on D12 and stained within three days.
94
miR-433 colony formation is presented as a relative ratio to pmiR-scr, and data are
95
expressed as the average ± SEM of biological duplicates. (C) CD34+ cells were stably
96
transduced with miRZIP-433 or miRZIP scrambled control lentivirus, and plated in
97
methylcellulose supporting myeloid colony formation. Three independent experiments
98
were performed, and colony number was assessed on day 14. miRZIP-433 colony
99
formation is presented as a relative ratio to miRZIP-scr, and data are expressed as the
100
mean ± SEM of biological triplicates (p<0.05). (D) CD34+ cells were stably transduced
101
with miRZIP-433 or scrambled control lentivirus, and plated in Megacult-C supporting
102
CFU-MK colony formation. Two independent experiments were performed. Slides were
103
fixed on day 12 and stained within three days. miRZIP-433 colony formation is
104
presented as a relative ratio to miRZIP-scr, and data are expressed as the average ±
105
SEM of biological duplicates.
106
107
Fig 9S. An independent experiment to confirm miR-433 silencing in ABM CD34+
108
promotes erythroid proliferation. CD34+ cells were stably transduced with miRZIP-
109
433 miRNA inhibitor or miRZIP scrambled control lentivirus, and cultured in erythroid
110
liquid culture for 9 days. The number of live cells in each sample was counted using
111
Countess Automated Cell Counter on days 3, 6 and 9, and are expressed in a
112
cumulative growth curve.
113
114
Table 7S. Genes deregulated in miR-433 overexpressing CD34+ cells and
115
predicted to be direct targets of miR-433 by miRecords (functional information
116
from GeneCards®).
117
118
Fig 10S. GBP2 Illumina array raw intensity.
119
120
Fig 11S. GBP2 overexpression and knockdown confirmation. (A) TF-1 were stably
121
transduced with GBP2 or empty vector control lentivirus, and cultured in 1U/ml EPO.
122
Total RNA was extracted on day 5 and the expression level of GBP2 was measured by
123
qRT-PCR. Average expression is expressed as the mean ± SEM of biological
124
triplicates. (B) TF-1 were stably transduced with a shRNA targeting GBP2 or scrambled
125
shRNA control lentivirus, and cultured in 1U/ml EPO. Total RNA was extracted on day
126
5 and the expression level of GBP2 was measured by qRT-PCR. Average expression
127
is expressed as the mean ± SEM of technical triplicates.
128
129
Fig 12S. MMP-9 expression in TF-1 cells with GBP2 overexpression or
130
knockdown. (A) Total RNA was extracted from TF-1 cells stably overexpressing GBP2
131
or empty vector.
132
expressed as the mean ± SEM of technical triplicates. (B) Total RNA was extracted
133
from TF-1 cells stably transduced with GBP2 shRNA or scrambled control. MMP-9
134
expression level was measured by qRT-PCR. Data is expressed as the mean ± SEM of
135
technical triplicates.
136
MMP-9 expression level was measured by qRT-PCR. Data is
137
Fig 13S. FACS analysis of TF-1 cells with GBP2 overexpression or knockdown.
138
(A) FACS analysis was performed using stably transduced TF-1 cells overexpressing
139
GBP2. Cells were cultured in 2ug/ml GMCSF as baseline for EPO differentiation
140
analysis. Surface markers CD71 (APC) and CD235a (PE) were measured. Data are
141
expressed in biological replicates. (B) FACS analysis was performed using stably
142
transduced TF-1 cells overexpressing GBP2, and cultured in 1U/ml EPO.
143
Differentiation was determined by measuring surface markers CD71 (APC) and
144
CD235a (PE) on day 6. Data are expressed in biological triplicates. (C) FACS analysis
145
was performed using TF-1 cells with reduced GBP2 expression, and cultured in 1U/ml
146
EPO. Differentiation was determined by measuring surface markers CD71 (APC) and
147
CD235a (PE) on day 6. Data are expressed in biological triplicates.
148
149
Fig 14S. A separate shRNA targeting GBP2 confirms that reduced GBP2 level
150
inhibits proliferation and accelerates erythroid differentiation in TF-1. (A) TF-1
151
were stably transduced with shRNA’ GBP2 or scrambled shRNA control lentivirus, and
152
cultured in 1U/ml EPO. Total RNA was extracted on day 5 and the expression level of
153
GBP2 was measured by qRT-PCR. Average expression is expressed as the mean ±
154
SEM of biological triplicates. (B) TF-1 cells stably transduced shRNA’ GBP2 or
155
scrambled control lentivirus were cultured in 1U/ml EPO for 6 days. The number of live
156
cells in each sample was counted using Countess Automated Cell Counter on days 3
157
and 6, and are expressed in a cumulative growth curve. Data are expressed as the
158
mean ± SEM of biological triplicates (*P<0.05). (C) FACS analysis was performed
159
using TF-1 cells with reduced GBP2 expression, and cultured in 1U/ml EPO.
160
Differentiation was determined by measuring surface markers CD71 (APC) and
161
CD235a (PE) on day 6. Data are expressed in biological triplicates.