1500.......1510........1520.................1530........
AAAAAUGUAGUUUACA
NOTCH1 rat
NOTCH1 mouse
UAAAGUGUAGUUUACA
NOTCH1 chimpanzee
AUAAAAUAGAGUGUAGUUUACA
NOTCH1 human
5’ AUAAAAUAGAGUGUAGUUUACAG...3’
|||||||
miR-30a
3'
GAAGGUCAGCUCCUACAAAUGU 5’
240........250.......260.......270........280...........290..
UUGGGGCCCC--UCGCU
NOTCH2.1 rat
UUGAGACCCC-UUGCUC
NOTCH2.1 mouse
CAAGCCUUGGGUCCAUGUUUACU
NOTCH2.1 chimpanzee
5’ CAAGCCUUGGGUCCAUGUUUACU. . .3’ NOTCH2.1 human
|||||||
3' GAAGGUCAGCUCCUA CAAAUGU
5’ miR-30a
......1980......1990..........2000................2010...
AUGGCAGU--------NOTCH2.2 rat
ACAGCAGU-CUA---UA
NOTCH2.2 mouse
NOTCH2.2 chimpanzee
GUUGGCAUAAUAGUUUACAAAU
5’
GUUGGCAUAAUAGUUUACAAAU 3'
NOTCH2.2 human
|||||||
3' GAAGGUCAGCUCCUACAAAUGU
5’ miR-30a
Supplementary Figure 1. MiR-30a binding sites in NOTCH1 and NOTCH2 3’UTRs. The miR-30a binding site in NOTCH1
(nucleotides 1520-1526 of the human 3’UTR sequence) is highly conserved (top panel), whereas the two NOTCH2 binding sites
(middle and bottom panels) are present only in primates (NOTCH2.1 at nucleotides 257-263 and NOTCH2.2 at nucleotides 19911997 of the human 3’UTR sequence). In addition to seed sequence (red font), all three sites show an additional complementary
nucleotide – seed match 7mer-1A for NOTCH1; 7mer-m8 and 7mer-1A for NOTCH2.1 and NOTCH2.2, respectively.
5
KOPTK1 miR-30a
KOPTK1 MSCV
DND41 miR-30a
0
DND41 MSCV
KOPTK1 miR-30a
KOPTK1 MSCV
DND41 miR-30a
DND41 MSCV
DHL7 miR-30a
DHL7 MSCV
DHL6 miR-30a
DHL6 MSCV
0
10
DHL7 miR-30a
2
15
DHL7 MSCV
4
20
DHL6 miR-30a
6
25
DHL6 MSCV
miR-30a relative expression
miR30a Delta CT values
8
Supplementary Figure 2. Expression of miR-30a in genetically modified DLBCL and T-ALL cell lines. Stable integration of
MSCV-30a constructs elevated the expression of this miRNA in all four cell line models created. In the left panel, the data are
displayed as Delta CT (miR-30a values normalized by control snoRNA U18 values) and, as expected, in all instances the DCT value
is lower (i.e., higher gene expression) for miR-30a expressing cells than for the isogenic controls containing an empty MSCV-eGFP
construct. In the right panel, we display the relative expression of miR-30a in each cell line, as they compare to their isogenic MSCV
control. All miRNA and snoRNA quantifications were performed with the stem-loop RT-PCR assay; data show are mean ± SD of
triplicates.
Sponge control
Sponge miR-30a
Luciferase activity
500000
P<0.05
400000
300000
200000
100000
0
NOTCH1 WT
NOTCH1 Mut
Supplementary Figure 3. Activity and specificity of miR-30a-directed sponge constructs. HEK-293 cells stably expressing the
miR-30a-directed sponge or the inert control sponge constructs were transiently transfected with the pmiR luciferase-NOTCH1
binding site reporter plasmid (WT or mutant), miR-30a synthetic oligonucleotides and the pCMVβ-gal plasmid (for normalization
purposes). Synthetic miR-30a oligos were significantly more effective in suppressing the luciferase activity of the NOTCH1-WT
construct in cells constitutively expressing an inert control sponge than in those expressing the miR-30a-directed sponge (bars on
the left). This result demonstrates activity, as the miR-30a sponge effectively binds to this mature miRNA, sequesters it away from
its target, thus limiting luciferase inhibition. Conversely, the luciferase activity of a NOTCH1 binding site mutant reporter was
indistinguishable in the cells expressing an inert control or the miR-30a directed sponge, demonstrating specificity. Data shown are
mean and ±SD of an assay performed in triplicate. Two biological replicates were completed for this assay. P <0.05 (two tailed
Student’s t test)
ns
1.0
0.5
*
-30e
P<0.05
ns
-30d
P<0.05
*
-30c
ns
*
1.5
1.0
0.5
-30b
-30c
-30d
-30e
24h
48h
-30b
-30e
-30d
-30c
-30b
-30e
-30d
0.0
-30c
0.0
-30b
miR-30
(relative expression)
1.5
DMSO
GSI
2.0
miR-30
(relative expression)
MSCV
ICN1
72h
Supplementary Figure 4. MiR-30 family expression following genetic and pharmacological modulation of NOTCH1. Left
panel. Stable expression of intracellular NOTCH1 (ICN1) in the DLBCL cell line SU-DHL7 led to significant downregulation of miR30c and 30d (p<0.05, two-tailed Student’s t-test). Right panel. Exposure of the T-ALL cell line KOPT- K1 to the gamma-secretase
inhibitor Compound E (100nM), resulted in significant de-repression of miR-30b, -30c, and 30e; *(p<0.05, two-tailed Student’s ttest), ns = not significant. Relative miRNA-30 expression data represent the mean ± SD of two independent biological replicates,
each performed in duplicate (four data points).
Cell number (X106)
A)
MSCV
miR-30a
6
Cell number (X106)
10
8
*
*
4
*
2
*
MSCV
miR-30a
8
*
6
4
*
*
2
0
0
0
1
2
3
4
0
Days
5
1
2
6
5
Cell number (X10 )
ctrl-sponge
miR-30a-sponge
6
Cell number (X106)
6
4
*
3
*
*
2
*
1
0
0
1
2
3
DND-41
3
4
Days
5
KOPT-K1
DND-41
B)
*
4
5
Days
*
ctrl-sponge
miR-30a-sponge
5
4
*
3
*
2
*
1
0
0
1
2
3
4
5
Days
KOPT-K1
Supplementary Figure 5. MiR-30a regulates grow rate of T-ALL cell lines. A) The growth pattern of two NOTCH-1 mutant TALL cell lines stably expressing miR-30a or an empty vector (MSCV) was monitored daily using an automated blue (TB) exclusion
assay. In both models, miR-30a expression significantly reduced cell proliferation (upper panels) (* p<0.01, two-tailed Student’s ttest). B) Functional inactivation of miR-30a with stable expression of a specific sponge construct in the same T-ALL cell lines,
significantly enhanced their growth rate (lower panels) (p<0.01, two-tailed Student’s t-test). All data are mean ± SD of an assay
performed in triplicate. Three biological replicates were performed for each assay, each time in triplicate.
80
P<0.01
P<0.01
NS
2.0
Cells in G0/G1 (%)
Apoptosis (fold increase)
2.5
1.5
1.0
0.5
P<0.01
60
40
20
miR30a
13.6%
SSC-A
Annexin V PE
30
a
iR
m
SC
V
M
30
a
G1: 55.97%
S: 17.15%
G2M: 22.6%
MSCV
miR30a
G1: 47.23%
S: 36.97%
G2M: 15.53%
G1: 58.00%
S: 30.14%
G2M: 11.47%
MSCV
miR30a
DND41
miR30a
4.5%
SSC-A
MSCV
KOPT-K1
G1: 54.39%
S: 19.89%
G2M: 23.30%
Annexin V PE
Annexin V PE
KOPTK1
DND-41
SSC-A
DND41
6.10%
SSC-A
MSCV
KOPT-K1
iR
M
DND-41
m
SC
V
iR
m
M
0
30
a
SC
V
30
a
iR
m
M
SC
V
0.0
9.0%
KOPTK1
Annexin V PE
Supplementary Figure 6. Cell cycle arrest and/or apoptosis associated with loss of fitness in miR-30a expressing T-ALL
cell lines. Expression of miR-30a significantly limited the growth rate of T-ALL cell lines, as determined by daily automated
monitoring with a trypan blue exclusion assay (Figure 4A). Cells collected at day 5 of these assays, were analyzed for apoptosis by
Annexin V staining (left panel) and their cell cycle profile determined by propidium iodide (PI) staining (right panel). Expression of
miR-30a significantly increased apoptotic rate in both models, while G0/G1 arrest was evident in the KOPT-K1. Apoptosis data are
represented as mean ±SD of the fold increase in apoptosis in the miR-30a expressing cells compared to MSCV controls. Cell
cycle data are displayed as the actual percentage of miR-30a or MSCV controls cells in G0/G1. These assays were all performed in
triplicate and three biological replicates completed. NS, non-significant; P <0.01 (two tailed Student’s t test). A representative
histogram of each assay/cell line is also shown
kD
kD SU-DHL7 OCI-Ly18
DND-41 KOPT-K1
34
34
Caspase-3
Caspase-3
26
26
26
26
Cleaved caspase-3
17
Cleaved caspase-3
17
β-actin
30a-spg
ctrl-spg
30a-spg
ctrl-spg
miR-30a
MSCV
miR-30a
MSCV
β-actin
Supplementary Figure 7. MiR-30a influences apoptosis in T-ALL and DLBCL cell models. Left panel. Ectopic
expression of miR-30a in the T-ALL cell lines DND-41 and KOPT-K1 increased the abundance of cleaved caspase3, in
association with the expected decrease in intact (inactive) caspase3. These data support the FACS-based Annexin V results
shown in Supplementary Figure 6. Right panel. Functional depletion of miR-30a with sponge constructs in the DLBCL cell
lines SU-DHL7 and OCI-Ly18, resulted in a decrease in the processing of caspase3, with accumulation of the intact (inactive)
enzyme (top) and marked decrease in the cleaved (active) caspase-3 isoform. These data further validate the FACS-based
Annexin V results shown in Figure 4D.