Supplemental Materials and methods - Genes &...

Supplemental Materials and methods

Cell lines and culture

Mel-STR and Mel-STV cell lines, immortalized primary human melanocytes expressing RasV12

or empty vector, respectively, were described previously (Gupta et al. 2005) and provided by

Robert Weinberg. 67NR cells, a BALB/C mouse-derived breast cancer cell line (Aslakson and

Miller 1992), were provided by Fred Miller and were grown in DMEM medium supplemented

with 5% fetal bovine serum, 5% newborn calf serum, 1X non-essential amino acids (Invitrogen),

L-glutamine (2 mM) and 1X penicillin/streptomycin (Invitrogen). To generate B16-F0 cell lines

expressing Bcl-2, B16-F0 cells were stably transduced with a Bcl-2 retroviral vector

(pME2PUROSRαBCL2; Innes et al. 1999), provided by Suzanne Cory, and puromycin selected

for 4 days.

Proliferation and apoptosis assays

The proliferation rate of the B16-F0 cells expressing a Gas1 shRNA or non-silencing control, or

B16-F10 cells ectopically expressing Gas1 or vector was determined using CyQUANT® Cell

Proliferation Assay Kit according to the manufacturer (Invitrogen). To evaluate apoptosis in the

above cell lines, protein extracts were prepared and the levels of cleaved and non-cleaved Parp-1

were analyzed by immunoblotting using an antibody against Parp-1 (BioMol).

Transwell/Boyden invasion assays

Invasion assays were performed using the CytoSelect 24-Well Cell Migration and Invasion

Assay as described by the manufacturer (Cell Biolabs). In brief, B16-F0 cells expressing a Gas1

shRNA or non-silencing control, or B16-F10 cells were placed in the upper compartment of the

Transwell/Boyden chamber and invasion as well as migration into the lower chamber was

measured 48hrs later. Invasion percentage was determined by dividing the number of cells that

invaded over the number of cells that migrated.

Shh signaling assays

B16-F0 cells were transduced with a Gas1 or non-silencing shRNA and 7 days later, cell extracts

were prepared and analyzed by immunoblotting using an antibody against Gli1 (Santa Cruz) or,

as a loading control, Actin (Sigma). For B16-F10 cells, extracts were prepared 7 days following

ectopic expression of Gas1.

Oncomine database searches

The Haqq (Haqq et al. 2005) and Smith (Smith et al. 2005) melanoma microarray datasets were

accessed using the Oncomine Cancer Profiling Database (www.oncomine.org). The Haqq

melanoma dataset includes 10 normal mole, 5 primary melanoma and 17 metastatic melanoma

samples; the Smith melanoma dataset includes 6 early stage (normal, benign nevi, melanoma in

situ) and 7 advanced stage (vertical growth phase melanoma, metastatic growth phase melanoma,

melanoma positive lymph nodes) samples. Histograms depicting gene expression in each sample,

as well as a Student’s t-test giving a p value for the comparison of candidate gene expression

between the groups, were obtained directly through the Oncomine 3.0 software. Analogous

approaches were used to analyze expression of candidate genes in other cancers.

Supplemental References

Aslakson, C.J. and Miller, F.R. 1992. Selective events in the metastatic process defined by

analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.

Cancer Res. 52: 1399-1405.

Gupta, P.B., Kuperwasser, C., Brunet, J.P., Ramaswamy, S., Kuo, W.L., Gray, J.W., Naber, S.P.,

and Weinberg, R.A. 2005. The melanocyte differentiation program predisposes to

metastasis after neoplastic transformation. Nat. Genet. 37: 1047-1054.

Haqq, C., Nosrati, M., Sudilovsky, D., Crothers, J., Khodabakhsh, D., Pulliam, B.L., Federman,

S., Miller, J.R., 3rd, Allen, R.E., Singer, M.I., Leong, S.P., Ljung, B.M., Sagebiel, R.W.,

and Kashani-Sabet, M. 2005. The gene expression signatures of melanoma progression.

Proc. Natl. Acad. Sci. USA 102: 6092-6097.

Innes, K.M., Szilvassy, S.J., Davidson, H.E., Gibson, L., Adams, J.M., and Cory, S. 1999.

Retroviral transduction of enriched hematopoietic stem cells allows lifelong Bcl-2

expression in multiple lineages but does not perturb hematopoiesis. Exp Hematol 27: 75-

87.

Smith, A.P., Hoek, K., and Becker, D. 2005. Whole-genome expression profiling of the

melanoma progression pathway reveals marked molecular differences between

nevi/melanoma in situ and advanced-stage melanomas. Cancer Biol. Ther. 4: 1018-1029.

Supplemental Table S1. Quantitation of satellite colony formation in the 3-D assay shown inSupplemental Figure S1

Gene Number of colonies Gene Number of coloniesB16-F0/NS -- Mipep +B16-F10 +++ Muc19 ++Acta2 ++ Nbeal2 +++Adamts16 +++ Nme7 +++Agl + N6amt1 ++Alg6 +++ Olfr198 +++Atg7 +++ Optc +++Baiap2 +++ Otud4 ++Bnip1 ++ Pclo +++B630019K06Rik +++ Pcsk6 +Cabyr +++ Pdyn +Cav2 + Phka1 +Ccdc39 +++ Ppp1r10 +++Ccna2 + Pramel3 +Cd5 ++ Prdm13 +++Cdc26 +++ Prrg3 +Centd1 +++ Ran +Cldnd2 + Rfx2 ++Col24a1 +++ Robo2 ++Cpa2 ++ Sccpdh +++Crkl +++ Setd2 ++Ctso + Slc9a3r2 +Dazl + Slc25a1 +Ddhd2 +++ Spc25 +Degs2 +++ Ss18l1 ++Dgkb ++ Thsd7b +++Dkc1 ++ Topbp1 ++Dmrta1 ++ Tomm70a +++Dnajb10 +++ Ttc17 +Dppa3 +++ Ubn1 +EG436563 + Ugt2b34 +++Fchsd1 + Uqcrc2 +++Fignl1 ++ Zadh1 +Gas1 +++ Zcchc9 +++Gimap6 + Zc3h13 +Gprasp1 + Zfp294 +++Hsd3b2 +++ 1500011H22Rik +Map2k2 ++ 4732496O08Rik +++Maz ++ 4931422A03Rik ++Mettl5 + 9630031F12Rik +

Satellite colonies were counted as scored as follows: – (0-10 colonies), + (11-25 colonies), ++ (26-50 colonies) or+++ (>50 colonies).

Supplemental Table S2. List of 78 candidate metastasis suppressor genes that tested positive in the 3-D cellculture assay.

Accession Gene Name

number symbol

NM_007392 Acta2 actin, alpha 2, smooth muscle, aorta

NM_172053 Adamts16 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1

motif, 16

NM_001081326 Agl amylo-1,6-glucosidase, 4-alpha-glucanotransferase

NM_001081264 Alg6 asparagine-linked glycosylation 6 homolog (yeast, alpha-1,3,-glucosyltransferase)

NM_028835 Atg7 autophagy-related 7 (yeast)

NM_130862 Baiap2 brain-specific angiogenesis inhibitor 1-asssociated protein 2

NM_172149 Bnip1 BCL2/adenovirus E1B interacting protein 1, NIP1

NM_175327 B630019K06Rik RIKEN cDNA B630019K06 gene

NM_027687 Cabyr calcium-binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2)

NM_016900 Cav2 caveolin 2

NM_026222 Ccdc39 coiled-coil domain containing 39

NM_009828 Ccna2 cyclin A2

NM_007650 Cd5 CD5 antigen

NM_139291 Cdc26 cell division cycle 26

AK122316 Centd1 centaurin, delta 1

NM_028849 Cldnd2 claudin domain containing 2

NM_027770 Col24a1 procollagen, type XXIV, alpha 1

NM_001024698 Cpa2 carboxypeptidase A2, pancreatic

NM_007764 Crkl v-crk sarcoma virus CT10 oncogene homolog (avian)-like

NM_177662 Ctso cathepsin O

NM_010021 Dazl deleted in azoospermia-like

AK173010 Ddhd2 DDHD domain containing 2

NM_027299 Degs2 degenerative spermatocyte homolog 2 (Drosophila), lipid desaturase

NM_178681 Dgkb diacylglycerol kinase, beta

NM_001030307 Dkc1 dyskeratosis congenita 1, dyskerin homolog (human)

NM_175647 Dmrta1 doublesex and mab-3 related transcription factor like family A1

NM_178055 Dnajb10 DnaJ (Hsp40) homolog, subfamily B, member 10

NM_139218 Dppa3 developmental pluripotency-associated 3

XM_489896 EG436563 predicted gene, EG436563

NM_175684 Fchsd1 FCH and double SH3 domain 1

NM_021891 Fignl1 fidgetin-like 1

NM_008086 Gas1 growth arrest specific 1

NM_153175 Gimap6 GTPase, IMAP family member 6

NM_026081 Gprasp1 G protein-coupled receptor associated sorting protein 1

NM_153193 Hsd3b2 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2

NM_023138 Map2k2 mitogen activated protein kinase kinase 2

NM_010772 Maz MYC-associated zinc finger protein (purine-binding transcription factor)

NM_029280 Mettl5 methyltransferase like 5

NM_027436 Mipep mitochondrial intermediate peptidase

NM_207243 Muc19 mucin 19

AK129158 Nbeal2 neurobeachin-like 2

NM_178071 Nme7 non-metastatic cells 7, protein expressed in

NM_026366 N6amt1 N-6 adenine-specific DNA methyltransferase 1 (putative)

NM_001011808 Olfr198 olfactory receptor 198

NM_054076 Optc opticin

NM_001081164 Otud4 OTU domain containing 4

NM_011995 Pclo piccolo (presynaptic cytomatrix protein)

BC037450 Pcsk6 proprotein convertase subtilisin/kexin type 6

NM_018863 Pdyn prodynorphin

NM_173021 Phka1 phosphorylase kinase alpha 1

NM_175934 Ppp1r10 protein phosphatase 1, regulatory subunit 10

NM_031390 Pramel3 preferentially expressed antigen in melanoma-like 3

NM_001080771 Prdm13 PR domain containing 13

NM_001081135 Prrg3 proline rich Gla (G-carboxyglutamic acid) 3 (transmembrane)

NM_009391 Ran RAN, member RAS oncogene family

NM_009056 Rfx2 regulatory factor X, 2 (influences HLA class II expression)

NM_175549 Robo2 roundabout homolog 2 (Drosophila)

NM_178653 Sccpdh saccharopine dehydrogenase (putative)

NM_001081340 Setd2 SET domain containing 2

NM_023055 Slc9a3r2 solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 2

NM_153150 Slc25a1 solute carrier family 25 (mitochondrial carrier, citrate transporter), member 1

NM_025565 Spc25 SPC25, NDC80 kinetochore complex component, homology (S. cerevisiae)

NM_178750 Ss18l1 synovial sarcoma translocation gene on chromosome 18-like 1

NM_172485 Thsd7b thrombospondin, type I, domain containing 7B

NM_176979 Topbp1 topoisomerase (DNA) II beta binding protein

NM_138599 Tomm70a translocase of outer mitochondrial membrane 70 homolog A (yeast)

NM_183106 Ttc17 tetratricopeptide repeat domain 17

NM_026666 Ubn1 ubinuclein 1

NM_153598 Ugt2b34 UDP glucuronosyltransferase 2 family, polypeptide B34

NM_025899 Uqcrc2 ubiquinol cytochrome c reductase core protein 2

NM_029880 Zadh1 zinc binding alcohol dehydrogenase, domain containing 1

NM_145453 Zcchc9 zinc finger, CCHC domain containing 9

NM_026083 Zc3h13 zinc finger CCCH type containing 13

NM_0010810686 Zfp294 zinc finger protein 294

NM_026883 1500011H22Rik RIKEN cDNA 1500011H22 gene

NM_172877 4732496O08Rik RIKEN cDNA 4732496O08 gene

AK076716 4931422A03Rik RIKEN cDNA 4931422A03 gene

NM_021416 9630031F12Rik RIKEN cDNA 9630031F12 gene

NOTE: Five of the genes (B630019K06RlK, Olfr835, Pramel3, Tdpoz2, 4931422A03RIK) lack obvious human homologues.

Supplemental Table S3. List of genes that are down-regulated in metastatic versus non-metastaticsamples, or in late (stages III and IV) relative to early (stage I) disease of multiple cancer types

Gene

mel

anom

a

brea

st

pros

tate

ovar

ian

liver

sarc

oma

colo

n

lung

blad

der

gastr

ic

head

-nec

k

sem

inom

a

Ewin

g’s s

arco

ma

cerv

ical

rena

l

ACTA2

ALG6

CCDC39

CCNA2

CTSO

CPA2

DPPA3

GAS1

PDYN

PHKA1

SETD2

SLC9A3R2

THSD7B

TOMM70A

ZNF294

indicates down-regulation in metastatic versus non-metastatic samples, or in late relative toearly stages of various human cancers. ZNF294 is the human homolog of mouse Zfp294.

Supplemental Table S4. shRNA source ID numbers and clone locations

Gene Source ID Clone Location

Acta2 V2MM_62388 SM2246-B-6V2MM_65336 SM2693-F-5

Alg6 V2MM_106772 SM2309-F-12V2MM_106773 SM2504-H-4

B630019K06Rik V2MM_225897 SM2483-E-7V2MM_88141 SM2138-E-6

Ccdc39 V2MM_64393 SM2006-G-6V2MM_74034 SM2215-F-10

Ccna2 V2MM_66832 SM2249-E-4V2MM_79073 SM2690-C-6

Cpa2 V2MM_108526 SM2498-H-11V2MM_206039 SM2505-G-12

Ctso V2MM_89746 SM2138-D-6V2MM_192286 SM2264-E-1

Dppa3 V2MM_41977 SM2596-H-1V2MM_52134 SM2442-G-8

EG436563 V2MM_211026 SM2635-A-1V2MM_214895 SM2368-H-1

Gas1 V2MM_77518 SM2002-B-1V2HS_114059 SH2395-E-1

Hsd3b2 V2MM_113015 SM2503-B-8V2MM_233631 SM2509-E-3

Olfr198 V2MM_206933 SM2332-E-9V2MM_140900 SM2053-B-9

Pdyn V2MM_14195 SM2388-C-5TRCN0000108487 AAN31-D-2

Phka1 V2MM_189266 SM2185-E-3V2MM_50701 SM2442-B-11

Prdm13 V2MM_233010 SM2499-E-9V2MM_106522 SM2502-F-3

Prrg3 V2MM_121756 SM2354-A-1V2MM_241522 SM2532-E-7

Slc9a3r2 V2MM_67362 SM2005-A-7V2MM_188689 SM2177-C-10

Setd2 V2MM_110951 SM2152-A-2V2MM_110955 SM2616-G-2

Thsd7b V2MM_203844 SM2492-C-10V2MM_204528 SM2507-C-2

Tomm70a V2MM_47658 SM2409-D-10V2MM_58390 SM2442-A-3

Zfp294 V2MM_102500 SM2493-H-6V2MM_206507 SM2492-C-1

4931422A03Rik V2MM_87414 SM2469-B-12V2MM_87411 SM2461-F-6

Supplemental Table S5. Primer sequences for quantitative real-time RT-PCR analyses

Gene Forward orreverse primer

Sequence (5’ 3’)

Acta2 forward TCCCTGGAGAAGAGCTACGAACTreverse AAGCGTTCGTTTCCAATGGT

Alg6 forward GCCTCGCCACATCCAGATAGreverse GCGCACAGCTAACCAGAGTAAA

B630019K06Rik forward TATGTTGGGGTTGGGTGAATreverse TTTCTAGCGCTGACAAAGCA

Ccdc39 forward CTGTTGCTGAGCGGAAAGTGreverse TGCCTTCGTGACGCTGGTA

Ccna2 forward AGGCCAGCTGAGCTTAAAGAAAreverse GATTCAAAACTGCCATCCATTG

Cpa2 forward GGACTCTGGGAAAAGCATTGACreverse CAAGAACTTGATCTCCCACAAATGT

Ctso forward CAGTTGAAGTTGGTGGCAGATTCreverse TGGCCCCTGAAGTTATATGCA

Dppa3 forward CTTTGTTGTCGGTGCTGAAAreverse TCCCGTTCAAACTCATTTCC

EG436563 forward TGGGTTTGATGAAGAAAGCAreverse TCCTTGGGAATTTTTGATCC

Gas1 (mouse) forward CCCTCTTCTGTGCGGTTTTCreverse CACCGTTCAGCACTCTGAGTC

GAS1 (human) forward TGGGACAGATAGAAGGGATGGTTreverse ACCGGCTACACCAAGTTGACTT

Hsd3b2 forward AAACACAGAAGTGGATGCTCACAreverse TTGCAGATCCCTTTAGCTCCTT

Olfr198 forward TCCTTCTTGCCATCCTGAAAAreverse GGGACGCACATACATGAGGAA

Pdyn forward TGGCAACGGAAAAGAATCTGAreverse TGTGCGGCTTCATCATTCA

Phka1 forward AGCCGCTGGAGGATGACAreverse ATGGTGTGCGAGATAGGGAAA

Prdm13 forward TCACTGCATAGCAAGCAAAACAGreverse TACTCAATGTGGCAAGGAGTGTTC

Prrg3 forward CCCTGAGCTCTCTCTCTCCAreverse AAGAGACACTGGCCTCCTCA

Slc9a3r2 forward TGGTGGCTCTGTGTGCTCATreverse CTGCCATCCTCATTGTCCTTCT

Setd2 forward CCTAGTTGTGAGCTGTTGGTGTAACreverse AGGTGCGCATGGGTCTGT

Thsd7b forward CCGGACTCGATTTATCATTATGCreverse TTGCCAAGGACCCAACTATAGC

Tomm70a forward CCTTCTTTCTTGGGGTAGGGreverse TGCAGCTTTCAGTGCAGTTT

Zfp294 forward AGCACCTGCAGTTTCTCTCCreverse GGTTTGGCTTTGACGACATT

4931422A03Rik forward GCACCGCCCCATTACTTGreverse CCCTCTGCTGCTCATCTTATCC

Supplemental Figure Legends

Supplemental Figure S1. Three-dimensional cell culture assays for each of the 78 B16-F0

knockdown cell lines. Collagen/MatrigelTM-embedded cells are shown in the center, surrounded

by migrating satellite colonies in the fibrin matrix. B16-F0 cells expressing a non-silencing (NS)

shRNA and B16-F10 cells are shown as controls.

Supplemental Figure S2. Analysis of target gene expression in the B16-F0 knockdown cell

lines. (A) Quantitative real-time RT-PCR (qRT-PCR) confirmed in all cases that expression of

the target gene was decreased in each B16-F0 KD cell line. Error bars indicate SEM (n=3). (B)

Immunoblot analysis for Gas1, Acta2 and Ccna2. Actin is shown as a loading control.

Supplemental Figure S3. Three-dimensional cell culture assays and target gene analysis for

each of the candidate 22 B16-F0 knockdown cell lines using a second, unrelated shRNA. For all

22 genes, a second, unrelated shRNA directed against the same target also resulted in increased

formation of satellite colonies in the three-dimensional assay (A) and knockdown of target gene

expression (B). Error bars indicate SD (n=3).

Supplemental Figure S4. Gas1 does not affect proliferation or apoptosis in cultured B16-F0 or

B16-F10 cells. (A) Proliferation of B16-F0 cells expressing a Gas1 or non-silencing (NS)

shRNA, and B16-F10 cells ectopically expressing Gas1 or empty vector was assessed at 24 and

48 hours. Error bars indicate SEM (n=3). (B) Levels of apoptosis in the indicated cultured cells

lines were determined by immunoblotting for Parp-1. Cleaved Parp-1, a marker of apoptosis, is

indicated by an arrow.

Supplemental Figure S5. Gas1 suppresses metastasis by promoting apoptosis of cells following

dissemination to secondary sites (24 hour time point). Quantitation of in vivo single-cell

fluorescence imaging of the lung 24 hours following tail vein injection of fluorescently labeled

B16-F0 cells stably expressing a Gas1 or NS shRNA. Error bars represent SEM (n=3 mice);

p=0.0027.

Supplemental Figure S6. Ectopic expression of the anti-apoptotic protein Bcl-2 increases

satellite colony formation in the 3-D cell culture assay. B16-F0 cells stably expressing Bcl-2 or

vector were grown for 8 days in the 3-D cell culture system. Satellite colonies were counted

under 100X magnification. Error bars indicate SEM (n=3).

Supplemental Figure S7. Knockdown of GAS1 in UACC-257 cells increases their ability to

form satellite colonies in the 3-D cell culture assay. Human melanoma UACC-257 cells stably

expressing a GAS1 or NS shRNA were grown for 8 days in the 3-D cell culture system. Error

bars indicate SEM (n=3).

Supplemental Figure S8. GAS1 expression is reduced in Mel-STR cells relative to Mel-STV

cells. qRT-PCR analysis of Gas1 expression in immortalized human melanocyte Mel-STV

(vector) cells relative to Mel-STR (Ras-expressing) cells. Error bars represent SD (n=3). The

results show that GAS1 expression is reduced ~6-fold in Mel-STR cells relative to Mel-STV

cells.

Supplemental Figure S9. Knockdown of Gas1 does not increase the invasion of B16-F0 cells.

Transwell/Boyden chamber assays comparing the invasion of B16-F0/Gas1 KD, B16-F0/NS, and

B16-F10 cells. Cells were placed in the upper compartment of the Transwell/Boyden chamber

and invasion and migration into the lower chamber was measured 48 hours later. Invasion

percentage was determined by dividing the number of cells that invaded by the number of cells

that migrated. Error bars indicate SEM (n=3).

Supplemental Figure S10. Gas1 does not affect expression of Gli1, a marker of Sonic

hedgehog signaling, in B16 mouse melanoma cells. Immunoblot analysis monitoring Gli1

expression in B16-F0 cells expressing a Gas1 or non-silencing (NS) shRNA, and in B16-F10

cells ectopically expressing Gas1 or empty vector. Actin was monitored as a loading control.

The results show that Gas1 knockdown in B16-F0 cells or Gas1 over-expression in B16-F10

cells does not affect Shh signaling.

Supplemental Figure S11. Down-regulation of ACTA2, CTSO, SLC9A3R2 and DPPA3 in

human metastatic melanoma cell lines and tissue samples. (A) ACTA2, CTSO and SLC9A3R2

expression in benign nevi (blue), primary melanoma (yellow) and metastatic melanoma (red).

(B) DPPA3 expression in advanced (red) versus early (blue) stage melanoma. Data were

obtained through the Oncomine database; a Student’s t-test, performed directly though the

Oncomine 3.0 software, showed the difference in gene expression between the samples was

significant.

Supplemental Figure S12. Knockdown of Gas1 in the mouse breast cancer cell line 67NR

increases lung metastasis. Left, mouse tail vein metastasis assay showing lungs from mice

injected with 67NR cells stably expressing a Gas1 or NS shRNA. Right, quantitation of lung

weight. In contrast to the experiments performed with B16 cells, in which the number of

metastases could be directly counted, here metastasis was quantified by weighing the lungs;

direct counting was not possible due to the large number of metastases, the large size of the

metastatic nodules, and the lack of pigmentation. Error bars represent SEM (67NR/NS, n=9

mice; 67NR/Gas1 KD, n=12 mice).

Acta2 Adamts16 Agl Alg6 Atg7 Baiap2 Bnip1 B630019K06Rik

Cabyr Cav2 Ccdc39 Cdc26 Centd1 Cldnd2Cd5

Dkc1

Col24a1 Cpa2 Crkl Ctso Dazl Ddhd2 Degs2 Dgkb

Gimap6

Nbeal2

Dmrta1 Dnajb10 Dppa3 Gas1

Gprasp1 Hsd3b2 Map2k2 Maz Mettl5 Mipep Muc19

Nme7

EG436563

Ccna2

Fchsd1 Fignl1

N6amt1 Olfr198 Optc Otud4 Pclo Pcsk6

Pdyn Phka1 Pramel3 Prdm13Ppp1r10 Prrg3 Ran Rfx2

Ss18l1 Thsd7b

Zadh1 Zcchc9

9630031F12Rik

1500011H22Rik

4732496O08Rik

4931422A03RikZc3h13 Zfp294

Uqcrc2Topbp1 Tomm70a Ttc17 Ubn1 Ugt2b34

Robo2 Sccpdh Setd2 Slc9a3r2 Slc25a1 Spc25

Supplemental Figure S1

NS B16-F10

14

18

12108

4

0

Fold

-kno

ckdo

wn

6

2

Acta2Alg6

B6300

19K06

Rik

Ccdc3

9

Ccna2

Ctso

Dppa3

Gas1

Hsd3b

2

4931

422A

03Rik

Olfr198Pdy

nPhk

a1Prrg

3Setd

2

Slc9a3

r2

EG4365

63

Thsd7

b1

Tomm70

a

Zfp294

Prdm13

Cpa2

16


B

A

Gas1 Acta2 Ccna2

NS NS NSGas1

Acta2

Ccna2

Gas1Actin

Acta2Actin

Ccna2Actin

shRNA:


Zfp294

Ctso

4931422A03Rik

Phka1

Ccna2Alg6 Cpa2B630019K06Rik Ccdc39Acta2

EG436563Dppa3 Olfr198Gas1 PdynHsd3b2

Prrg3 Thsd7b Tomm70aSetd2 Slc9a3r2Prdm13

A

B

14

18

12108

4

0

Fold

-kno

ckdo

wn

6

2

Acta2Alg6

B6300

19K06

Rik

Ccdc3

9

Ccna2

Ctso

Dppa3

Hsd3b

2

Tomm70

a

Olfr198Pdy

nPhk

a1Prrg

3Setd

2

Slc9a3

r2Gas

1

Tdpoz

2Ths

d Zfp

Prdm13

Cpa2

16

NS

4931

422A

03Rik


Cel

l num

ber

0

A

2000400060008000

1000012000

1600014000

18000B16-F0/NSB16-F0/Gas1 KDB16-F10+vectorB16-F10+Gas1

Time (hours)

Immunoblot

Parp-1

Actin

B16-F

0/NS

B16-F

0/Gas

1 KD

B16-F

10+v

ector

B16-F

10+G

as1

B

240 48

Num

ber o

f lab

eled

cells

rem

aini

ng

16

10

0hr: 24

1214


8

246

B16-F0/NSB16-F0/Gas1 KD


Num

ber o

f col

onie

s

16

101214

8

246

Bcl-2

vecto

r0

18

B16-F0


Num

ber o

f col

onie

s

40

253035

20

51015

0

45

UACC-257

GAS1NSshRNA:

GAS1

Rat

io o

f nor

mal

ized

mR

NA

leve

ls (M

el-S

TV/M

el-S

TR)

9

45

3210

678


0

% In

vasi

on


B16-F

0/NS

B16-F

0/Gas

1 KD

B16-F

10

51015202530354045

Gli1

Actin

B16-F

0/NS

B16-F

0/Gas

1 KD

B16-F

10+v

ector

B16-F

10+G

as1


A ACTA2

T-test: 4.363 P-value: 1.4E-4

-3.0

2.01.00.0

-2.0-1.0

-4.0

CTSO

T-test: 3.026 P-value: 0.006

3.02.01.00.0

-2.0-1.0

BSLC9A3R2

T-test: 4.827 P-value: 3.7E-4

3.02.01.00.0

-2.0-1.0

DPPA3

T-test: 2.95 P-value: 0.014-2.5

-0.5

-1.5

0.0

-2.0

-1.0

0.51.0

Nor

mal

ized

expr

essi

on u

nits

Nor

mal

ized

expr

essi

on u

nits

Nor

mal

ized

expr

essi

on u

nits

Nor

mal

ized

expr

essi

on u

nits


https://mail.umassmed.edu/Judy.Mondor/Inbox/No%20Subject-2734.EML/1_multipart_xF8FF_1_MSG.9:3.doc/C58EA28C-18C0-4a97-9AF2-036E93DDAFB3/MSG.9_3.doc?attach=1

67NR/NS

67NR/Gas1 KD W

eigh

t per

lung

(g)

1.2

0.8

0.4

0

1.4

1.0

0.6

0.2

Gas1NSshRNA:


Supplemental Materials and methods - Genes &...

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