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Supplemental Information
Figure S1. MDA separation from coculture with hMSCs using MACS.
Flow cytometry analysis on (a) coculture of hMSC and MDA cells and on (b)
MDA cells after negative sorting of CD90 positive hMSCs using MACS
(Magnetic activated cell sorting).
a. after coculture
63%
99.5% 0.5%
SS Lin
37%
b. MDA after MACS sorting
CD90PE
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Figure S2. Expression of IFN-β in hMSCs after stimulation of poly (dA:dT)
and TNF-α. Quantitative RT-PCR for IFN-β from MSCs treated with or
without TNF-α (10 ng/ml) or poly (dA:dT) for 24 hrs. MSCs increase IFN-β1
mRNA expression upon poly (dA:dT) stimulation in dose dependent manner.
400
RQ for IFN-beta
350
300
250
200
150
100
50
0
0
MSC
1
5
10
20
50
100 200 500
MSC + TNF-α + Poly (dA:dT) (ng/mL)
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Figure S3. Assessment of the efficacy of AIM2 siRNA and IFIH1 siRNA in
hMSCs. Quantitative RT-PCR assays for AIM2 (A) and IFIH1 (B) in the
RQ for Aim2
A
600
B
250
500
RQ for IFIH1
hMSCs that were isolated from the experiment in Figure 4D.
200
400
300
200
100
150
100
50
0
0
control
scr
Aim2
hMSCs siRNA siRNA
hMSCs hMSCs
fr CCT fr CCT
control
scr
IFIH1
hMSCs siRNA siRNA
hMSCs hMSCs
fr CCT fr CCT
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Figure S4. IFN-β upregulates TRAIL expression in hMSCs during
coculture with MDA cells. (A~D) Real-time RT-PCR assays for AIM2, IFIH1,
IRF7 and TRAIL in hMSCs treated with different concentrations of rhIFN-β.
Values are mean ± S.D. for triplicate of assay. (E~H) Real-time RT-PCR
assays for AIM2, IFIH1, IRF7 and TRAIL in hMSCs treated with supernatant
from act hMSC-MDA coculture (CCT sup) that was treated with different
concentrations of IFN-β neutralizing antibody (α-IFN-β). Values are mean ±
S.D. for triplicate of assay.
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Figure S5. Kaplan-Meier survival analysis of the innate sensors and
related genes in breast cancer. (A-C) Kaplan-Meier survival analysis of the
all
0 20 40 60 80 100
RFS (%)
A
0 20 40 60 80 100
AIM2 (A), IFIH1 (B), TLR3 (C) in all and ER-positive breast cancer.
P=0.00088
ER+
P=0.9
AIM2 low
AIM2 high
0 20 40 60 80 100 120
Time (months)
AIM2 low
AIM2 high
0 20 40 60 80 100 120
Time (months)
IFIH1 low
IFIH1 high
0 20 40 60 80 100 120
Time (months)
RFS (%)
C
0 20 40 60 80 100
Number at risk
low 1782 1581 1362 1158 890 600 388
high 1772 1492 1182 930 722 470 296
0 20 40 60 80 100
P=0.002
P=0.22
IFIH1 low
IFIH1 high
0 20 40 60 80 100 120
Time (months)
Number at risk
low 901 835 732 634 507 370 243
high 901 821 682 540 430 279 174
P=8.4e-13
TLR3 low
TLR3 high
0 20 40 60 80 100 120
Time (months)
0 20 40 60 80 100
RFS (%)
B
0 20 40 60 80 100
Number at risk
Number at risk
low 1779 1522 1218 959 747 477 311 low 903 824 674 535 421 257 172
high 1775 1551 1326 1119 865 593 373 high 899 832 740 639 516 382 245
P=0.0024
TLR3 low
TLR3 high
0 20 40 60 80 100 120
Time (months)
Number at risk
Number at risk
low 1781 1479 1202 989 761 506 313 low 906 821 694 589 471 313 187
high 1773 1594 1342 1099 851 564 371 high 896 835 720 585 466 336 230
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List of primers
Taqman Gene Expression Assay ID
Name
(Life Technologies)
Human-specific GAPDH
Hs00266705_g1
Human TRAIL (TNFSF10)
Hs00921974_m1
Human TRAIL receptor 1
(DR4; TNFRSF10A)
Human TRAIL receptor 2
(DR5; TNFRSF10B)
Hs00269492_m1
Hs00366278_m1
Human AIM2
Hs00915710_m1
Human IFIH1
Hs01070332_m1
Human Fas
Hs00236330_m1
Human Fas Ligand
(FASLG)
Hs00181225_m1
List of Antibodies
Antigen
Clone
Company
Dilution
TRAIL
Rabbit mAb (C92B9)
Cell Signaling Technology
1:1000
PKC-α
Rabbit mAb
Cell Signaling Technology
1:1000
Cell Signaling Technology
1:1000
Sigma Aldrich
1:25000
Phospho- Rabbit mAb
PKC-α
(Thr638/641)
β-actin
Clone AC-15
For secondary antibodies, HRP-linked anti-rabbit IgG or anti-mouse IgG (Cell
Signaling Technology; 1:2,000) were used.
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Preparation of TRAIL reporter construct.
Genomic DNA isolated from hMSCs (QIAamp Tissue Kit; QIAGEN) was used
as the template with primers for the human TRAIL promoter (Figure 1 for
sequences for primers), using the Expand High Fidelity PCR System (Roche
Molecular Biochemicals) following the manufacturer’s instruction. The
amplified DNA fragments (1183bp) were subcloned into the KpnI and NheI
sites in the pGL4 plasmid containing the luciferase reporter gene (pGL4-17;
Promega). The PCR construct was amplified between the highlighted area in
TRAIL promoter sequence.
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
aaaatttgaaaatattttcttaaatgtagactcatttacagatagaaggcaagggcagga
agtgatggtgaccagcggtgcctgaatgaactcaggaatgtaactgtagatctagggtcc
caaactttaggtttcaaaggatctcttggagtacttgctgaaaaatgtaggttcctaagt
ccactgccagaaactctgactcagtgggtcaagaatggaataactaaacaatggccccat
gcagtggttcatgcctgtaatcccagcacgttgggaggttgaagcaagaggatcacttga
ggtcaggagttcgagaccagcctggcctacatgataaaaccccatctctactaaaaatac
aaaaaaattagctgggcatggtggcatgcacctgtaatcccagctacttgggaggctgag
gcaggagaattgcttgaatctgggaggtggaggttgtagtgggccgagattgtgccattg
caccactgcactccagcctgggcgataaagtgagattctgtcaaaaaaataaataaatac
atgaaagagagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaagaaaga
aagaaagaaagaaggaaagaaggaaagaatagaaaagaaaagaaagaaagggaggaagaa
aaggaaagaaagaaatgctgaataagatatagagacacatacagctgggccagcttatga
catctgatagtggggagatttggggctgggtcctgaatctgagggtaattaactccctgt
aacttcttttcctaatctgtaaaaggatagtgacagcgagacattgtgatggggttaata
ttttggaaaacatccacatgtttttttcctttgcctttctgagtgtgtcaactacttcct
acctgtccagcctaacacacaggcatattgtcttggtagggatggagatctgagaaggag
attagaatttgtgtctgaaggtttgcaaagaggaagaagtcgtcaatatttagattctga
cattcaagatggaattatgtagcaagaccattgctatgagacagtatttctattttcctt
tatccactcccaccctgccctcttcccaccctcacagtagcatgagaaaaaccacatatg
gaagtttcaggtcataaaaattatcttataatttagaaaacaggccttgtgcctatgaca
gccaggccatgaggcttagagctctgtggtagaatgaggatatgttagggaaaagcaaag
aaaatccctcccctccttggctgaggacattatcaaaaggagagcaagaaagagaagaga
gaaatgggcttgaggtgagtgcagataaggggtgcatggatcctgagggcaaggagagga
gcttctttcagtttccctcctttccaacgactactttgagacaagagctgtccctgggca
gtaggaagggggagggacagttgcaggttcaatagatgtgggtggggccaaggccacaga
acccagaaaaacaactcattcgctttcatttcctcactgactataaaagaatagagaagg
aagggcttcagtgaccggctgcctggctgacttacagcagtcagactctgacaggatcat
ggctatgatggaggtccaggggggacccagcctgggacagacctgcgtgctga
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Stable transfection
MDA cells were transfected with TRAIL-pGL4 reporter using Lipofectamine
2000 (Life Technologies) as manufacturer’s instruction. After 72 hours of
transfection, MDA cells were treated with 1000 μg/mL neomycin (Sigma) for
the first week and 500 μg/mL neomycin in DMEM containing 10 % FBS for 2
more weeks. The media was changed every 3rd day and the cells were
passaged in 1:4 upon 80 % confluency. The transfected cells were than
plated <100 cells/cm2 and cultured until the cells start to form colonies. The
colonies were then isolated and cultured until they reach sufficient number.
Expanded clone of MDA cells were tested for luciferase activity and the clone
with the strongest activity was selected for further experiments
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