Supplementary Information
S1A.Densitometric analysis of MCM7 protein expression in different phases of cell cycle in
K562 cells shows the mean ±s.e.m of three individual experiments (*p<0.05). Beta actin was
used as a loading control.
S1B.Densitometric analysis of MCM7 protein expression in different phases of cell cycle in
HEL cells shows the mean±s.e.m of three individual experiments (*p<0.05). Beta actin was
used as a loading control.
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S1C. Relative gene expression of MCM 7 at different phases of cell cycle normalised against
HPRT1 as seen by qRT-PCR in CMK cells. Data represents the mean ± s.e.m of three
independent experiments (*p<0.05, n=3).
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S2A.Densitometric analysis of MCM7 protein expression normalised against coomassie
stained membrane in HEL cells treated with TPA for the indicated time points in days. Data
shows the mean±s.e.m of three individual experiments (*p<0.05, **p<0.01).
S2B. MCM7 promoter activity during megakaryopoiesis in HEL cell as seen by Dual
luciferase assay. Data represents the mean ± s.e.m of three independent experiments
(*p<0.05, n=3).
S2C.Relative gene expression of miR-17-92 cluster and miR-106a in ≥8N population when
compared to 2N-4N population as seen by qRT-PCR in HEL cells. The relative expressions
were normalised against HPRT1. Data represents the mean ± s.e.m of three independent
experiments (*p<0.05, n=3).
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S2D. In vitro megakaryocyte culture from cord blood derived CD34+ cells were characterized
at day 0 and day 10 with anti CD61 and anti CD42b antibodies. The unilineage
megakaryocyte cultures on day 10 were stained with vibrant orange. The polyploid
population was sorted in a flow cytometer.
S2E.Relative gene expression of MCM7 and miR-106b-25 cluster in in vitro megakaryocyte
culture from cord blood derived CD34+ cells: ≥8N population was compared to 2N-4N
population and analyzed by qRT-PCR in day 10. The relative expressions were normalised
against HPRT1. Data represents mean ± s.e.m of three independent experiments (*p<0.05,
n=3).
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S2F.Relative gene expression of MCM7 and miR-106b-25 cluster in higher ploidy population
compared to lower ploidy in CMK cells. The relative expressions were normalised against
HPRT1. Data represents the mean ± s.e.m of three independent experiments (*p<0.05, n=3).
S2G.Western blot of whole cell lysate of CMK cells treated with TPA showing the
expression of MCM7. Densitometric analysis of MCM7 protein expression normalised
against coomassie stained membrane. Data shows the mean ± s.e.m of three individual
experiments (*p<0.05,).
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S3A. Relative fold change in gene expression of MCM7 and miR-106b-25 cluster in mock
and sh-UPF1 transfected TPA treated HEL cell as seen by qRT-PCR. The relative
expressions were normalised against HPRT1. Data represents the mean ± s.e.m of three
independent experiments (*p<0.01, n=3).
S3B. Relative fold change of ratio between the amounts of larger transcript and both
transcript as compared to TPA untreated HEL cells. Data represents the mean ± s.e.m of three
independent experiments (*p<0.05, n=3).
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S4A. CD61 marker analysis in mock and miR-106b transfected HEL cells.
S4B. CD42b marker analysis in mock and miR-106b transfected HEL cells.
HEL
Transfected
with
Mock
Median intensity value of
CD61 (± SE)
8.03(±0.0208)
miR-106b
Transfected
with
P-value
Mock
8.02(±0.0176)
Median intensity value of
CD42b (± SE)
193.74(±2.0069)
miR-106b
195.84(±1.29)
0.0004
P-value
0.0032
0.0027
0.0001
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S4C. CD61marker analysis in mock and miR-93 transfected HEL cells.
HEL
Transfected with
Median intensity value of CD61 (± SE)
P-value
Mock
11.14(±0.0577)
0.0032
miR-93
11.75(±0.3724)
0.0375
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S5A.Relative gene expression of megakaryopoietic lineage specific transcription factors in
miR-25 transfected K562 cells as compared to mock transfected cells. Data represents the
mean ± s.e.m of three independent experiments (*p<0.05, n=3) normalised against HPRT1.
S5B. Relative gene expression of megakaryopoietic lineage specific transcription factors in
anti-miR-25 transfected HEL cells as compared to scrambled siRNA transfected cells. Data
represents the mean ± s.e.m of three independent experiments (*p<0.05, n=3) normalised
against HPRT1.
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S5C.Densitometric analysis of PTEN, phospho-Akt protein expression in mock and miR-25
containing vector transfected HEL and K562 cell line shows the mean±s.e.m of three
individual experiments (p<0.03 for K562, p<0.01 for HEL).
S5D.Densitometric analysis of PTEN protein expression in scrambled siRNA and anti miR25 transfected HEL and K562 cell line shows the mean ±s.e.m of three individual
experiments (p<0.03).
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S5E.Densitometric analysis of PTEN protein expression in HEL cells treated with TPA for
the indicated time points in days shows the mean ± s.e.m of three individual
experiments(p<0.01).
S6A. Relative gene expression of miR-25 in miR-25 transfected HEL and K562 cells as
compared to mock transfected cells. Data represents the mean ± s.e.m of three independent
experiments (*p<0.05, n=3) normalised against HPRT1.
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S6B. Relative gene expression of miR-25 in miR-25 nucleofected CD34+ cells as compared
to mock transfected cells. Data represents the mean ± s.e.m of three independent experiments
(*p<0.05, n=3) normalised against HPRT1.
S6C. CD42b expression in HEL cells due to transfection of ‘mock’, ‘miR-25’, ‘PTEN’,
‘PTEN+miR-25’.
ANOVA result:
F value (156.6410); Fctitical value (4.0661).
As the computed F exceeds the critical F, so it is inferred that there is a significant change in
CD42b marker expression between the groups, and the four groups differ significantly
(P<0.000001).
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