Supplement Data
Supplemental Table 1. Stem-loop structure oligonucleotide sequences.
tgctgttgacagtgagcgaccagatacctgcaccaccttatagtgaagccacagatgtataaggt MMP2 ggtgcaggtatctgggtgcctactgcctcgga tgctgttgacagtgagcgccgatgctgccatttctaataatagtgaagccacagatgtattatta MMP3 gaaatggcagcatcgatgcctactgcctcgga tgctgttgacagtgagcgcgcaaggttatcccaaggatattagtgaagccacagatgtaatatcc MMP8 ttgggataaccttgcatgcctactgcctcgga tgctgttgacagtgagcgccgacatagacggcatccagtatagtgaagccacagatgtatactgg MMP9 atgccgtctatgtcgttgcctactgcctcgga tgctgttgacagtgagcgacgagcctgaatttcatttgattagtgaagccacagatgtaatcaaa MMP10 tgaaattcaggctcggtgcctactgcctcgga tgctgttgacagtgagcgagcaatatttcagctaccaatatagtgaagccacagatgtatattgg MMP11 tagctgaaatattgcctgcctactgcctcgga tgctgttgacagtgagcgccgcgggaatcctgaaggagaatagtgaagccacagatgtattctcc MMP13 ttcaggattcccgcgatgcctactgcctcgga tgctgttgacagtgagcgcggctgacatcatgatcttatttagtgaagccacagatgtaaataag MMP14 atcatgatgtcagccttgcctactgcctcgga tgctgttgacagtgagcgcggccacaccttcttcttccaatagtgaagccacagatgtattggaa MMP15 gaagaaggtgtggccttgcctactgcctcgga tgctgttgacagtgagcgaggcaaacgtgatgtggatatatagtgaagccacagatgtatatatc MMP16 cacatcacgtttgccgtgcctactgcctcgga tgctgttgacagtgagcgcaggaaggatattacacctatttagtgaagccacagatgtaaatagg MMP24 tgtaatatccttcctttgcctactgcctcgga tgctgttgacagtgagcgaccgactcctgctatacctttatagtgaagccacagatgtataaagg ADAM8 tatagcaggagtcggctgcctactgcctcgg tgctgttgacagtgagcgcgccatttcactctgtcatttatagtgaagccacagatgtataaatg ADAM10 acagagtgaaatggcatgcctactgcctcgga tgctgttgacagtgagcgccgacatcctctccttagctaatagtgaagccacagatgtattagct ADAM17 aaggagaggatgtcgttgcctactgcctcgga MOCK tgctgttgacagtgagcgcggcttcagactcattattatatagtgaagccacagatgtatataat *(clock) aatgagtctgaagccatgcctactgcctcgga * The shRNA targeting the clock gene (circadian locomoter output cycles kaput) was used as the mock shRNA in all experiments to test the target specificity.
Supplemental Table 2. Primers used for RT-PCR for mouse MMPs (mMMPs) and human MMPs (hMMPs).
Forward Reverse Size (bp) mMMP-2 caccaccacaactgaaccac ctcagaagagcccgcagtag 199 mMMP-3 cactccctgggactctacca tggtgatgtctcaggttcca 201 mMMP-7 gatgatgaggacgcaggagt cagcgtgttcctctttccat 195 mMMP-8 agctgtcaagggctgaagtg taccttggcctggttgaaag 201 mMMP-9 caccaccacaactgaaccac ctcagaagagcccgcagtag 199 mMMP-10 cactccctgggtctctttca tttgtctggggtctcaggtc 201 mMMP-11 tgagccttagcccagatgac aagagctctcctcggatggt 199 mMMP-12 cccagaggtcaagatggatg aagtctccgtgagctccaaa 202 mMMP-13 tttattgttgctgcccatga ctctggtgttttgggatgct 198 mMMP-20 cgacaatgctgagaagtgga gggtccgtataatgcttgga 202 mMMP-23 cgtcccagtgacctcaagat gccaaacacctttcttccaa 199 mMMP-14 (MT1- ccctaggcctggaacattct tttgggcttatctgggacag 200 MMP) mMMP-15(MT2- cctcccacgacaagaactgt ggaaagaagctgcagaatgc 200 MMP) mMMP-16 (MT3- ggagacagttccccatttga cgttggaatgttccagtcct 196 MMP) mMMP-17 (MT4- caggaggaactgtccaaagc ccctccaagaaaggttcctc 202 MMP) mMMP-24 (MT5- gacgcagcctatgaaagagc gtaccgttcgcctttgaaga 201 MMP) mMMP-25(MT6- ccatcagcaacacaggtgac cgcagtgacaatcacagctt 201 MMP) mMMP-26 atgcatgctctgctacacca cacattgctccagatggaaa 276 hMMP-14 ctctcttctggatgcccaat acctccgtctcctcctcagt 300
Supplemental Table 3. Primers used for RT-PCR for mouse ADAMs (mADAMs) and human ADAMs (hADAMs).
Forward Reverse Size (bp) mADAM 2 tgagcatatcggggctga agcatggggctagcgtat 397 mADAM 8 tgtcctggagggaacagaa aaccggttgacatctggaa 400 mADAM 9 catgaattggggcataac ctcactggtcttccctct 399 mADAM 10 agcaacatctggggacaa aaagttgggcttgggatc 398 mADAM 12 ccttaagatgaccaagta cattcagacagccccctt 401 mADAM 15 acaagcatcttaggcgttg ttgacaacagggtccatca 400 mADAM 17 tgagcgattttgggatttc gtccttctcaaatccgtca 405 mADAM 19 tgctcagctaatcacggg gaagacggtgaagaatgt 402 mADAM 33 gtgattgctgtgcgaggt gtgccgcacatagtgact 398 hADAM 10 tccggaatccgtaacatcag agtttgtccccagatgttgc 450 hADAM 17 tcattgaccagctgagcatc gagtctgtgctggggtcttc 400
Supplemental figure legends
Figure S1. Suppression of the expression of ADAM 10 or ADAM 17, but not MMPs, inhibits MICA shedding in Myc-CaP-MICA cells. A Real-time RT-PCR showing shRNA
suppression of MMP and ADAM expression in Myc-CaP-MICA cells. B. Degree of MICA
shedding in Myc-CaP-MICA cells with the suppression of indicated MMP or ADAM expression.
** p < 0.01 when compared to cells expressing mock shRNA.
Figure S2. MMP14 and ADAMs independently regulate MICA shedding in TRAMP-C2-
MICA cells. A and B, real-time RT-PCR (A) and flow cytometry (B) demonstrating that the
expression of ADAM 10 was not affected by suppressing MMP14 expression in TRAMP-C2
cells. The anti-ADAM 10 monoclonal antibody MAB946 (R&D Systems) was used for flow
cytometry analyses. Filled grey profiles, control rat IgG staining. C and D, real-time RT-PCR (C)
and Western blotting (D) demonstrating that the expression of ADAM 17 was not affected by
suppressing MMP14 expression in TRAMP-C2 cells. The anti-human and mouse ADAM 17
antibody PX084A (Cellsciences) was used for Western blot analyses. E and F, real-time RT-
PCR (E) and Western Blotting (F) demonstrating that suppression of ADAM 10 or ADAM 17
expression has no effect on MMP14 expression at mRNA or protein level. G and H, co-silencing
MMP 14 and ADAM 10 or ADAM 17 generates an additive effect in inhibiting MICA shedding.
Co-silencing was done by sequential transducing MMP14-shRNA cells with ADAM 10 or
ADAM 17 lentivirus. ** p < 0.01 and * p < 0.05 when compared to cells expressing mock
shRNA. Data represent results from five independent experiments.
Figure S3. Peptide sequence comparison of human MMP14 (hMMP-14) and mouse
MMP14 (mMMP-14).
Figure S4. Real-time RT-PCR showing relative MMP14 (A), ADAM 17 (B), and ADAM 10
(C) expression in representative human and mouse tumor cell lines. M12 and PC-3 are human prostate cancer cell lines. MCF-7 is a human breast cancer cell line. Primers used for human and mouse cell lines are listed in Supplemental Tables 2 and 3. Note different scales of relative expression in each plot.
Figure S5. Overexpression of MMP14 in MCF-7 cells increases MICA shedding and reduces surface MICA expression. A. Real-time RT-PCR showing overexpression of MMP14 in MCF-7 cells. B. ELISA assay demonstrating that overexpression of MMP14 markedly increases MICA shedding in MCF-7 cells. **, p < 0.01 when compared to control MCF-7 cells.
C. Flow cytometry demonstrating that overexpression of MMP14 reduced surface MICA expression on MCF-7 cells. Filled grey profiles, control rat IgG staining.
Figure S6. Histograms of flow cytometry showing MIC (A and B) expression on MCF-7 and M12 cells. Cells were stained with control mouse IgG, anti-MIC(A/B) mAb 6D4.6, or anti-
MICB MAB1599 (R&D systems) followed by PE-conjugated goat anti-mouse IgG. Grey filled profiles, control mouse IgG staining. Filled black profiles, mAb 6D4.6 staining. Open black line profiles, MAB1599 staining. Data show that neither MCF-7 nor M12 cells express MICB.
Figure S1
A mock shRNA Specific shRNA 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Relative expression Relative 0.0 3 P9 P11 17 MP M M10 M MMP2 M M A MM DA AD A B 0.3
0.2
0.1 ** ** MICA shedding MICA (SN : LY) 0 1 5 -9 1 1 17 ock P M10 M shRNA: m P A MMP MM DA MM MMP24 AD A
Figure S2
A B
ADAM10 TC2-MICA TC2-MICA 1.5 TC2-MICA- shRNA MMP14 TC2-MICA- 1.0 shRNA ADAM10
0.5 ** Relative expression 0 1 2 3 4 0 1 2 3 4 0.0 10 10 10 10 10 10 10 10 10 10 shRNA: mock ADAM10 MMP14 ADAM 10
C D ADAM17 1.2 17 AM P14 shRNA: D 1.0 mock M A M 0.8 0.6 ADAM17 0.4 ** Relative expression 0.2 ERK 0.0 shRNA: MOCK ADAM17 MMP14
F E 4 10 MMP14 1 M 17 shRNA: ck P M o DA 1.2 MM A DA 1.0 m A 0.8 0.6 MMP14 0.4 ** 0.2
Relative expression 0.0 ERK 4 ck 1 10 17 shRNA: P mo AM AM MM D D A A
G H 1.4 MMP14 1.2 1.2 1.0 * * 1.0 0.8 0.8 0.6 0.6 0.4 ** ** ** ** ** 0.4 ** 0.2 MICA shedding MICA(LY:SN) Relative expression 0.2 0.0 0.0 10 17 7 10 k 10 17 shRNA: AM AM MOCK D D shRNA: P14 M MMP14 A A moc M AM ADAM M DA D / A A /ADAM1 4 4 / ADAM17 14 1 1 P P MM MMP MMP14/ADAM10 MM
Figure S3
hMMP-14: 1 MSPAPRPPRCLLLPLLTLGTALASLGSAQSSSFSPEAWLQQYGYLPPGDLRTHTQRSPQS mMMP-14: 1 MSPAPRPSRSLLLPLLTLGTALASLGWAQGSNFSPEAWLQQYGYLPPGDLRTHTQRSPQS ******* * **************** ** * **************************** +- hMMP-14: 61 LSAAIAAMQKFYGLQVTGKADADTMKAMRRPRCGVPDKFGAEIKANVRRKRYAIQGLKWQ mMMP-14: 61 LSAAIAAMQKFYGLQVTGKADLATMMAMRRPRCGVPDKFGTEIKANVRRKRYAIQGLKWQ ********************* ** ************** ******************* hMMP-14: 121 HNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIF mMMP-14: 121 HNEITFCIQNYTPKVGEYATFEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIL ******************** ************************************** hMMP-14: 181 FAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHE mMMP-14: 181 FAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVQNEDLNGNDIFLVAVHE ******************************************* **************** hMMP-14: 241 LGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGFPTKMPPQPRTT mMMP-14: 241 LGHALGLEHSNDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGSKSGSPTKMPPQPRTT ********** ********************************* ** *********** hMMP-14: 301 SRPSVPDKPKNPTYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVMDGYPMPIGQF mMMP-14: 301 SRPSVPDKPKNPAYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVMDGYPMPIGQF ************ *********************************************** hMMP-14: 361 WRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYPKHIKELGRGLPTDKIDAALF mMMP-14: 361 WRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYPKHIKELGRGLPTDKIDAALF ************************************************************ hMMP-14: 421 WMPNGKTYFFRGNKYYRFNEELRAVDSEYPKNIKVWEGIPESPRGSFMGSDEVFTYFYKG mMMP-14: 421 WMPNGKTYFFRGNKYYRFNEEFRAVDSEYPKNIKVWEGIPESPRGSFMGSDEVFTYFYKG ********************* ************************************** hMMP-14: 481 NKYWKFNNQKLKVEPGYPKSALRDWMGCPSGGRPDEGTEEETEVIIIEVDEEGGGAVSAA mMMP-14: 481 NKYWKFNNQKLKVEPGYPKSALRDWMGCPSGGRPDEGTEEETEVIIIEVDEEGSGAVSAA ***************************************************** ****** hMMP-14: 541 AVVLPVLLLLLVLAVGLAVFFFRRHGTPRRLLYCQRSLLDKV mMMP-14: 541 AVVLPVLLLLLVLAVGLAVFFFRRHGTPKRLLYCQRSLLDKV **************************** *************
Figure S4
A. MMP14 MMP14 1.5 1.5
1.0 1.0
0.5 0.5
0.0 Relative expression Relative 0.0 TC2 MyC expression Relative M12 PC3 MCF7
B. 5.0 ADAM17 ADAM17 1.0 4.0 3.0 0.5 2.0 1.0 0.0
0.0 expression Relative
Relative expression Relative M12 PC3 MCF7 TC2 MyC
C. ADAM10 4.0 ADAM10 2.0 3.0 1.5 2.0 1.0 1.0 0.5 0.0 0.0 Relative expression Relative
TC2 MyC expression Relative M12 PC3 MCF7
Figure S5
AB C MMP14 MCF-7-MMP14 1.0 ** 2.0 ** 0.8 MCF-7 0.6 1.5 0.4 1.0 Relative Relative expression 0.2 0.5
0.0 MICA shedding MICA(SN : LY) 0 1 2 3 4 0.0 10 10 10 10 10 MCF-7 MCF-7- MCF-7 MCF-7- MMP14 MMP14 MICA
Figure S6
MCF-7 M12
MICA MICB MICA MICB Relative numberRelative Relative numberRelative
0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 Fluorescence Fluorescence