Supplementry Informtion Kim et l. Supplementry Mterils nd Methods Gene expression nlysis y Affymetrix microrry Totl RNAs were isolted from MDA-MB-231 cells expressing or using the RNesy mini kit (Qigen). Two independent iologicl replictes were ssyed for ech smple. The microrrys were performed following the Affymetrix stndrd protocol s descried previously 1. Using normlized log2 intensities, we identified DBC1-depedent genes y compring the differentil expression etween MDA-MB-231/ nd MDA-MB-231/ smples. The cutoff for differentilly expressed genes ws fold chnge of 1.5 or greter for the entire dtsets. The dt hve een deposited in the Gene Expression Omnius (GEO) dtse, www.nci.nlm.nih.gov/geo (ccession no. GSE5477). Plsmids nd ntiodies cdna encoding humn PEA3 (Open Biosystems) ws cloned into psg5.ha, pcdna3.1-v5/his, pgex-4t-1, nd pcdf PylT-1. cdnas encoding DBC1 nd SIRT1 were cloned into pgex-4t-1. packrs-3 nd pcdf PylT-1 vectors were kindly provided y Json W Chin (MRC Lortory for Moleculr Biology, Cmridge, UK) nd used for generting recominnt site-specificlly cetylted proteins in cteri. pcdf PylT-1-PEA3 constructs crrying mer muttions were generted with the QuikChnge site-directed mutgenesis kit (Strtgene). To generte the 5xEBS-TA-LUC reporter plsmid, oligonucleotides contining five repetitive copies of EBSs (5 -GCAGGAAGCA-3 ) were cloned into pta-luc vector (Clontech). The promoter regions of (nucleotides -255 to 7 reltive to trnscription strt site) nd (nucleotides -59 to 21 reltive to trnscription strt site) genes were mplified from humn genomic DNA nd cloned into pgl3-bsic vector (Promeg). The following plsmids were descried previously: psg5.ha-dbc1, psg5.flag-dbc1, S/FLAG/SBP- SIRT1, HA-p3, phr.cmv.puro.sin8-, plko.1-#3, nd phr.cmv.flag-luc.ires- Hygro 1-3. The lentivirl plko.1-puro vectors (plko.1-#5 nd plko.1-#8) contining the shrna ginst DBC1/CCAR2 (TRCN53724 nd TRCN53727) were purchsed from Sigm-Aldrich. Lentivirl prticles were generted s descried previously 3. psg5.ha-dbc1-r#3 encodes wild-type DBC1 ut contins silent muttions in codons 561 563 to mke it resistnt to 1
#3; the QuikChnge site-directed mutgenesis kit (Strtgene) ws used to generte the muttions with primers 5 -ATAAGATGCTACTAGtCTCCTGAAAAGGTCG-3 (forwrd) nd 5 - CGACCTTTTCAGGtAGCTtAGTAGCATCTTAT-3 (reverse) (muttions re shown in lowercse). The following ntiodies were used in this study: nti-pea3 ntiody 16X (113X), nti-dbc1 ntiody T-19, nti-p3 ntiody C-2, nti-rna Polymerse II ntiody 8WG16, nti- ntiody (SB12e), nd nti-tuulin ntiody TU-2 (Snt Cruz Biotechnology); nti- ntiody 274 (Acm); nti-flag ntiody M2 (Sigm); nti-v5 ntiody R96-25 (Invitrogen); nti-ha ntiody 3F1 (Roche); nti-dbc1 ntiody A3-434A, nti-sirt1 ntiody A3-688A, nd nti-s-tg ntiody A19-135A (Bethyl Lortories); nti-cetyl lysine ntiody #9441 (Cell Signling); nti- ETV4 ntiody M1 (Anov); nti-gapdh ntiody LF-PA18 (AFrontier). Protein-protein interction ssys nd immunolot For GST pull-down ssys, in vitro trnslted or purified recominnt proteins were incuted with immoilized GST-fusion proteins. Bound proteins were nlyzed y immunolot with nti-ha or nti-s- Tg ntiodies. For CoIP ssys, 293T or MDA-MB-231 cell extrcts were immunoprecipitted y specific ntiodies or control IgG nd protein G Dyneds (Invitrogen) s indicted in figure legends. In some experiments, conventionl horserdish peroxidse (HRP)-conjugted nti-mouse IgG ws sustituted with Mouse TrueBlot ULTRA: Anti-Mouse Ig HRP (Rocklnd) to eliminte interference of signl detection y the IgG hevy chins. Electrophoretic moility shift ssys (EMSAs) EMSAs were performed using DIG gel shift kit (Roche) s descried previously 4. Purified recominnt PEA3 proteins were incuted with digoxigenin (DIG)-end-leled doule-strnded oligonucleotide contining PEA3 consensus sequence (5 -GGCTAGTGAGCAGGAAGTAGGGAGAG-3 [sense] nd 5 -CTCTCCCTACTTCCTGCTCACTAGCC-3 [nti-sense]) t room temperture for 3 min. DNA protein complexes were resolved on 6% polycrylmide gels in.5x TBE uffer nd detected y chemiluminescence using DIG detection kits (Roche). Limited proteolytic digestion ssys Prtil proteolytic digests were performed y dding trypsin (Sigm) or Arg-C (Promeg) to unmodified nd site-specificlly cetylted PEA3. Digestions were crried out t room temperture for 1 min 2
(trypsin) or 1 hr (Arg-C). C-terminlly S-tgged frgments were detected y immunolot with nti-s- Tg ntiody. Acetyltion nd decetyltion ssys 293T cells were trnsfected with expression plsmids for PEA3, p3, SIRT1, nd DBC1 s indicted in the figure legends. After 36 hr of trnsfection, the cells were treted with.5 mm TSA (Sigm) for 12 hr nd then lysed in FLAG lysis uffer 2 supplemented with.5 μm TSA nd protese inhiitor cocktil (Roche). PEA3 ws immunoprecipitted with nti-pea3 ntiody, nd cetyltion levels were determined y immunolot with nti-cetyl lysine ntiody. For in vitro decetyltion ssys, sitespecificlly cetylted PEA3 ws incuted with purified recominnt GST-SIRT1 nd GST-DBC1 nd 1 mm NAD in SIRT1 rection uffer for 3 hr t 3 C, s descried previously 2. MTT nd flow cytometric nlysis MDA-MB-231, BT-2, nd SK-BR-3 cells infected with lentiviruses expressing or were plted t density of 2x1 4 cells/well in 24-well pltes. Cell prolifertion ws determined y MTT ssys (Promeg). For cell cycle nlysis, MDA-MB-231 cells were plted in 6-well pltes, synchronized y serum strvtion for 48 hr, nd then restimulted to enter the cell cycle y ddition of 1% FBS for 24 hr. Flow cytometric nlysis of cell cycle distriution ws performed s descried previously 2, 5. Colony Formtion, Scrtch, Migrtion, Invsion Assys For colony formtion ssy, MDA-MB-231 cells were plted t density of 1x1 3 cells/well in 6-well pltes nd grown until colonies ppered. Colonies were stined with crystl violet nd photogrphed s descried previously 1, 5. The stined cells were soluilized in 1% SDS, nd sornce ws mesured t 57 nm. For scrtch migrtion ssy, cell-free re (scrtch) ws creted y using 2 µl yellow tip on confluent cell monolyers. Photogrphs were tken t 24 hr. Wound closure ws determined using the initil nd finl wound res during the experiments s descried previously 5. Two-chmer migrtion nd invsion ssys were performed s descried previously 1, 5 using Trnswell inserts (Costr) coted with fironectin (Sigm) or with Mtrigel (BD Biosciences)/fironectin. Xenogrfts 3
MDA-MB-231 cells (3 1 6 cells) infected with lentiviruses expressing or were suspended in 1 μl Mtrigel/PBS (5:5 mixture) nd injected sucutneously into the right flnk of 6- week-old femle thymic BALB/c nu/nu mice (Orient Bio, Kore). Ech experimentl group contined ten mice. Tumors were mesured every week using digitl cliper nd the volumes were clculted s descried previously 1, 5. For in vivo ioluminescence imging, mice were nesthetized nd given 15 μg/g of D-luciferin (Xenogen) in PBS y intrperitonel injection. Fifteen minutes fter injection, ioluminescence ws imged with the IVIS Spectrum Imging System (Xenogen). The ioluminescence intensity ws expressed s photon flux (photons/sec/cm 2 /Sterdin). Xenogrft tumors were collected nd stored s FFPE (formlin-fixed prffin-emedded) tissues for further nlysis. RNAs from FFPE xenogrft tumors were extrcted using the RNesy FFPE kit (Qigen) followed y qrt-pcr nlysis to determine the expression levels of DBC1 nd PEA3 trget genes. ER-negtive rest tumor smples, tissue microrry (TMA), nd immunohistochemistry (IHC) TMA sections mounted on slides were deprffinized with xylene, rehydrted in seril dilutions of lcohol, nd immersed in peroxidse-locking solution (Dko) to quench endogenous peroxidse ctivity. For ntigen retrievl, sections were microwved in Trget Retrievl Solution (Dko) for 15 min. Sections were incuted with nti-dbc1 ntiodies for 3 hr, wshed with TBS-T, nd then incuted with horserdish peroxidse-lelled secondry nti-rit ntiody (EnVision Detection System, Dko) for 3 min. DAB ws used s chromogen. Digitl imges of tissue sections were cptured using the Aperio ScnScope XT slide scnner (Aperio Technologies). The scoring method of Sinicrope et l 6 ws used to evlute oth the intensity of immunohistochemicl stining nd the proportion of the stined epithelil cells. The stining intensity ws further clssified s follows: 1 (wek), 2 (moderte), nd 3 (strong). The positive cells were quntified s percentge of the totl numer of epithelil cells nd were ssigned to one of the following five ctegories: (<5%), 1 (5%-25%), 2 (26%-5%), 3 (51%- 75%), nd 4 (>75%). The percentge of positivity of the tumor cells nd the stining intensities were then multiplied to generte n IHC score (~12) for ech tumor specimen. An IHC score of more thn 8 ws regrded s high level of expression. Sttisticl nlysis Sttisticl significnce for qpcr, MTT, colony formtion, migrtion, invsion, nd xenogrft experiments ws estimted y unpired, two-tiled Student s t-test. p vlues re stted in the figure 4
legends. Sttisticl nlysis for IHC results ws performed using the SPSS 16. (SPSS Inc.). Kpln Meier plots nd log rnk tests were used for relpse-free survivl nlysis. Relpse-free survivl ws defined s the time from the dte of dignosis to the dte of documented relpse, including locoregionl recurrence nd distnt metstsis. p vlues <.5 were considered to e sttisticlly significnt nd ll sttisticl tests were two-sided. Primer sets used in this study qrt-pcr ws performed using following forwrd (F) nd reverse (R) primers: β-ctin, 5 -CCA CAC TGT GCC CAT CTA CG-3 (F) nd 5 -AGG ATC TTC ATG AGG TAG TCA GTC AG-3 (R);DBC1, 5 -AAG GTG CAA ACG CTC TCC AAC CAG-3 (F) nd 5 -GGA TGT TTG GAA GAG ACT CAG AG-3 (R);, 5 -GAG CAA ACA CAT CTG ACC TAC AGG A-3 (F) nd 5 -TTG TCC CGA TGA TCT CCC CTG ACA-3 (R);, 5 -CTG AGA AGC ATG ACG GAC AAG TAC-3 (F) nd 5 -GTA GAT GAC ATG GAC TGC CTT GCA-3 (R); PDGFA, 5 -CTG CAA GAC CAG GAC GGT CAT TTA-3 (F) nd 5 -GCT TCT TCC TGA CGT ATT CCA CCT-3 (R); GPR16, 5 -CCA GCC ATC TAC CAA AGC CTG-3 (F) nd 5 -GCC AGT AAC TCT GAA TGC TGA CA-3 (R); LPCAT2, 5 - GGG ATG GTG TTC GTA AGC ATT TGG-3 (F) nd 5 -CAT CTA TTG CGA GTT CCT CAA AAG-3 (R); IL8, 5 -TCT GCA GCT CTG TGT GAA GG-3 (F) nd 5 -ACT TCT CCA CAA CCC TCT GC-3 (R); BMP4, 5 -CAC TGG TCC CTG GGA TGT TC-3 (F) nd 5 -GAT CCA CAG CAC TGG TCT TGA CTA-3 (R); TGFBR2, 5 -CAT CTG TGA GAA GCC ACA GG-3 (F) nd 5 -TGC ACT CAT CAG AGC TAC AGG-3 (R); NRG1, 5 -ATG TGT CTT CAG AGT CTC CCA T-3 (F) nd 5 -TGG ACG TAC TGT AGA AGC TGG-3 (R); EGLN3, 5 -ATT CAT AGC AGA TGT GGA GCC-3 (F) nd 5 -TCA GCA TCA AAG TAC CAG ACA G-3 (R); CST1, 5 -GGT GGC ATC TAT AAC GCA GAC-3 (F) nd 5 -CTG CAG TTC TGG CTG TTC ATG-3 (R); TIMP3, 5 -ACG CTG GTC TAC ACC ATC AAG C-3 (F) nd 5 -CCG AAA TTG GAG AGC ATG TCG-3 (R); TLR4, 5 -GGC AGC TAT AGC TTC TTC AGT TTC-3 (F) nd 5 -GCC ACC AGC TTC TGT AAA CTT GAT-3 (R); PECAM1, 5 - CAA AGA CAA CCC CAC TGA AGA C-3 (F) nd 5 - CGC AAT GAT CAA GAG AGC AAT G-3 (R); SIRT1, 5 -CAT AGC CTT GTC AGA TAA GGA AGG-3 (F) nd 5 -CTC CTC GTA CAG CTT CAC AGT CAA-3 (R). ChIP qpcr ws performed using following primers (nucleotides reltive to trnscription strt site): promoter (-116~96), 5 -CTT GTT TGA AGT TAA TCA TGA CAT-3 (F) nd 5 -AGC CTC TTG CTG CTC CAA TAT C-3 (R); promoter (-148~-839), 5 -CTG ATT CGT GCC AAA GCT TGT CCT-3 (F) nd 5 -GAA CTG CGT GCC ACG CAC TGT TCT-3 (R); 5
GREB1-17 k upstrem, 5 -TAT TCC AGT GGC TGT CTT TGC-3 (F) nd 5 -AGG GGT CCA CAG GAC ATG A-3 (R). sirnas used in this study sidbc1#1 2 ; sidbc1#2 2 ; sins 3 ; sisirt1#2, 5 -GAA GUU GAC CUC CUC AUU GdTdT-3 (sense) nd 5 -CAA UGA GGA GGU CAA CUU CdTdT (nti-sense). Supplementry References 1. Seo WY, Jeong BC, Yu EJ, Kim HJ, Kim SH, Lim JE, et l. CCAR1 promotes chromtin loding of ndrogen receptor (AR) trnscription complex y stilizing the ssocition etween AR nd GATA2. Nucleic Acids Res 213; 41: 8526-8536. 2. Yu EJ, Kim SH, Heo K, Ou CY, Stllcup MR, Kim JH. Reciprocl roles of DBC1 nd SIRT1 in regulting estrogen receptor ctivity nd co-ctivtor synergy. Nucleic Acids Res 211; 39: 6932-6943. 3. Kim JH, Yng CK, Heo K, Roeder RG, An W, Stllcup MR. CCAR1, key regultor of meditor complex recruitment to nucler receptor trnscription complexes. Mol Cell 28; 31: 51-519. 4. Kim JH, Lee MH, Kim BJ, Hn SJ, Kim HY, Stllcup MR. Role of sprtte 351 in trnsctivtion nd ctive conformtion of estrogen receptor lph. J Mol Endocrinol 25; 35: 449-464. 5. Yu EJ, Kim SH, Kim MJ, Seo WY, Song KA, Kng MS, et l. SUMOyltion of ZFP282 potentites its positive effect on estrogen signling in rest tumorigenesis. Oncogene 213; 32: 416-4168. 6. Sinicrope FA, Run SB, Clery KR, Stephens LC, Lee JJ, Levin B. cl-2 nd p53 oncoprotein expression during colorectl tumorigenesis. Cncer Res 1995; 55: 237-241. 6
Supplementry Figure S1. Het mp digrm of DBC1-dependent genes in MDA-MB-231 cells. () Het mp of downregulted genes y DBC1 shrna. () Het mp of upregulted genes y DBC1 shrna. Fold log 2-3 3 Numer of overlpping genes 14 12 1 8 6 4 2 (35.9%) Cell communiction (GO:7154) (5.8%) (13.9%) Cell cycle (GO:749) Cell dhesion (GO:7155) (44.4%) Metolic process (GO:8152) (19.7%) Developmentl process (GO:3252) (22.4%) (13.5%) Immune system process (GO:2376) Trnsport (GO:681) (43.6%) Cellulr process (GO:9987) (14.3%) System process (GO:38) (29.7%) Others Supplementry Figure S2. Ontology nlyses nd functionl chrcteriztion of differentilly expressed overlpping genes. PANTHER (http://www.pntherd.org) th ws used to serch for over-represented Biologicl Process ctegories mong the differentilly expressed overlpping genes. 7
Reltive e mrna levels Reltive mrna levels.4 EGLN3.3 CST1.45.3.2.3.2.1.1.15 TLR4.4.15 PECAM1.3.1.2.1.55 TIMP3 DBC1 Tuulin Supplementry Figure S3. Vlidtion of suset of DBC1-regulted genes. () Totl RNAs from MDA-MB-231 cells infected with lentiviruses expressing or #3 were exmined y qrt-pcr nlysis with primers specific for the indicted mrnas. Results shown were normlized to β-ctin mrna levels nd re mens±sd (n=3) 3). () Protein levels were monitored y immunolot using the indicted ntiodies. Reltive mrn NA levels NA levels.18.12.6..9.6 DBC1.1.8.66.4.2 DBC1.12.8.5.4.33.2.1.12.8 Reltive mr.3.4.4 Supplementry Figure S4. DBC1 is required for nd expression in ERnegtive rest cncer cells. Totl RNAs from SK-BR-3 () ndbt-2() cells infected with lentiviruses expressing or #3 were exmined y qrt-pcr nlysis with primers specific for the indicted mrnas. Results shown were normlized to β-ctin mrna levels nd re mens±sd (n=3). 8
DBC1 GAPDH Reltive mrna levels DBC1.4.12.15.3.2.1.8.1.4.5 c Asornce t 57 nm 1.2.8.4 d 12 6 # of cells per field18 e 12 8 4 # of cells per field16 Supplementry Figure S5. DBC1 is required for PEA3 trget gene expression nd for cell prolifertion, migrtion, nd invsion of MDA-MB-231 cells. To rule out the possile off-trget effects, we used two dditionl shrnas (#5 nd #8) trgeting different regions of DBC1 mrna. ( nd ) ) DBC1 is required for the expression of nd in MDA-MB-231 cells. MDA-MB-231 cells were infected with lentiviruses expressing, #5 or #8. Protein levels were monitored y immunolot using the indicted ntiodies (). Totl RNA ws exmined y qrt-pcr nlysis with primers specific for the indicted mrnas (). Results shown were normlized to β-ctin mrna levels nd re mens±sd (n=3). (c, d, nde) Cell prolifertion, migrtion, nd invsion ssys. MDA-MB- 231 cells were infected with lentiviruses expressing, #5 or #8. Cell prolifertion ws determined y MTT ssy (c). Migrtion (d) nd invsion (e) ssys were performed using Trnswell chmers without or with Mtrigel. P-vlue ws determined y Student s t-test (P <.5 nd P <.1). 9
HA HA-DBC1-R#3 -DBC1 -HA -GAPDH Reltive mrna levels.2 15.15.1.5 HA HA-DBC1-R#3.5.4 #3.3.2.1 #3 c d e A sornce t 57 nm.6.4.2 HA HA-DBC1-R#3 #3 # of cells per field 6 4 2 HA HA-DBC1-R#3 #3 field12 # of cells per 8 4 HA HA-DBC1-R#3 #3 Supplementry Figure S6. Rescue of DBC1 shrna-induced defects in trnscriptionl ctivtion, cell prolifertion, migrtion, nd invsion. To exclude the possile off-trget effects cused y DBC1 RNAi, DBC1 expression ws recovered y expression of shrna-resistnt DBC1 in DBC1-depleted MDA-MB-231 cells. MDA-MB-231 cells infected with lentiviruses expressing #3 were trnsfected with either psg5.ha or psg5.ha-dbc1-r#3 expressing #3-resistnt DBC1. Protein levels were monitored y immunolot using the indicted ntiodies (). The indicted mrna levels were determined y qrt PCR (). Cell viility ws determined y MTT ssy (c). Migrtion (d) nd invsion (e) ssys were performed using Trnswell chmers without or with Mtrigel. P-vlue ws determined y Student s t-test (P <.5 nd P <.1). 1
IgG -DBC1 SK-BR-3 BT-2 BT-549 BC1 BC1 BC1 IP -PEA3 (113X) -ETV4 (M1) -DBC1 IP IgG -D IgG -D IgG -D -PEA3 (113X) -DBC1 Input -PEA3 (113X) -DBC1 Input -PEA3 (113X) -DBC1 c IP IgG -FLAG -HA (PEA3) -FLAG (DBC1)( ) d e 1 34 PEA3 AD ETS NTD 34 484 CTD Input -HA (PEA3) -FLAG (DBC1) HA-DBC1 Supplementry Figure S7. DBC1 intercts with PEA3. () Whole cell lystes of MDA-MB- 231 cells were immunoprecipitted with norml IgG or nti-dbc1 ntiody. The immunoprecipittes were nlyzed y immunolot with the indicted ntiodies. Coimmunoprecipitted PEA3 ws detected with two different PEA3 ntiodies (nti-pea3 113X, Snt Cruz; nti-etv4/pea3 M1, Anov), nd Truelot ULTRA ws used s the secondry ntiody to reduce the ckground due to the IgG hevy chin. Arrowheds indicte coimmunoprecipitted PEA3, nd sterisks represent mouse IgG hevy chin. () Whole cell lystes of SK-BR-3, BT-2, nd BT-549 were immunoprecipitted with norml IgG or nti- DBC1 ntiody. The immunoprecipittes were nlyzed y immunolot with the indicted ntiodies. Coimmunoprecipitted PEA3 ws detected with TrueBlot ULTRA to reduce the ckground due to the IgG hevy chin. Arrowheds indicte coimmunoprecipitted PEA3, nd sterisks represent mouse IgG hevy chin. (c) 293T cells were trnsfected with psg5.ha- PEA3 nd psg5.flag-dbc1. Cell lystes were immunoprecipitted with nti-flag ntiody or norml IgG. Input nd immunoprecipitted proteins were nlyzed y immunolot with the indicted ntiodies. (d) Schemtic representtion of full-length PEA3 nd deletion mutnts tested in GST pull-down ssys. AD, ctivtion domin; NTD, N-terminl domin; CTD, C-terminl domin. (e) DBC1 intercts with the N-terminl domin of PEA3. GST pull- downssyswereperformedusinginvitrotrnslteddbc1ndgst-pea3ntdorgst- PEA3 CTD. Bound proteins were nlyzed y immunolot with nti-ha ntiody. 11
IP Input IgG -V5 -FLAG (SIRT1) -V5 (PEA3) -FLAG (SIRT1) -V5 (PEA3) IP Input PEA3-V5 HA-p3 FLAG-SIRT1 -Ac K (Ac PEA3) -V5 (PEA3) -V5 (PEA3) -HA (p3) -FLAG (SIRT1) c -PEA3 -SIRT1 -DBC1 -Tuulin Input IgG -SIRT1 IP Supplementry Figure S8. SIRT1 intercts with nd decetyltes PEA3, nd DBC1 locks the interction etween PEA3 nd SIRT1. () 293T cells were trnsfected with S/FLAG/SBP- SIRT1 nd pcdna3.1-pea3-v5/his. Cell lystes were immunoprecipitted with nti-v5 ntiody or norml IgG. Input nd immunoprecipitted proteins were nlyzed y immunolot with the indicted ntiodies. () SIRT1 decetyltes PEA3. 293T cells were trnsfected with expression vectors s indicted. Cell extrcts were immunoprecipitted with nti-v5 ntiody, nd levels of cetylted PEA3 were determined y immunolot using nti-cetyl lysine (Ac K) ntiody. Input nd immunoprecipitted proteins were nlyzed y immunolot with the indicted ntiodies. (c) 293T cells were infected with lentiviruses expressing or #3. Cell lystes were immunoprecipitted with nti-dbc1 ntiody or norml IgG. The immunoprecipittes were nlyzed y immunolot with the indicted ntiodies. Arrowheds indicte coimmunoprecipitted PEA3, nd sterisks represent mouse IgG hevy chin. sins s sisirt1 s SIRT1 GAPDH Reltive mr RNA levels.8.6 4.4.2 SIRT1.24.5.4.16.3.8.2.1 sins sisirt1 sins sisirt1 sins sisirt1 c - EX-527 SIRT1 GAPDH d A levels Reltive mrn.32.24.16.8 Ex-527 -.5.4.3.2.1 Supplementry Figure S9. SIRT1 negtively regultes the expression of nd. ( nd ) MDA-MB-231 cells were trnsfected with SIRT1 sirna or non-specific (NS) sirna, s indicted. 72 hr fter trnsfection, protein levels were monitored y immunolot using the indicted ntiodies (). Totl RNA ws exmined y rel-time qrt-pcr nlysis with primers specific for the indicted mrnas (). Results shown were normlized to -ctin mrna levels nd re mens±sd (n=3). (c nd d) MDA-MB-231 cells were treted with 5 M EX-527 for 24 hr. Immunolot (c) nd qrt-pcr nlyses were performed s descried in nd. 12 -
1% 8% 6% 4% 2% % Serum (-) Serum () G2/M S G1 Supplementry Figure S1. Effect of DBC1 depletion on cell cycle progression. MDA-MB- 231 cells infected with lentiviruses expressing or #3 were precultured in serumfree medium for 48 hr, then treted with 1% FBS for 24 hr, nd sujected to FACS nlysis. Serum tretment stimulted cell cycle progression of MDA-MB-231/ cells, ut serum stimultion of cell cycle progression ws ttenuted y DBC1 depletion. Reltive mr RNA levels.2.15.1.5 DBC1.2.15.1.5.8.6.4.2 Supplementry Figure S11. qrt-pcr nlysis of nd expression in two pirs of MDA-MB-231 xenogrft tumors infected with lentiviruses expressing or #3. Totl RNAs were purified from formlin-fixed prffin-emedded (FFPE) xenogrft tumor smples nd nlyzed y qrt-pcr for DBC1,, nd expression. Results shown were normlized to -ctin mrna levels nd re mens±sd (n=3). Asterisks indicte sttisticlly significnt differences (P <.5 nd P <.1; Student t-test). 13
Rdvnyi Brest Curtis Brest TCGA Brest p=4.34x1-6 p=2.16x1-4 p=1.3x1-1 Norml Invsive crcinom Norml Invsive crcinom Norml Invsive crcinom Supplementry Figure S12. Bioinformtics nlysis of DBC1 mrna expression in humn invsive rest crcinom smples using dtse t ONCOMINE (http://www.oncomine.org). P-vlue ws determined y Student s t-test. Low expression High expression IHC score : -3 IHC score : 4-7 IHC score : 8 Supplementry Figure S13. Representtive imges showing different levels of DBC1 immunostining in ER-negtive rest crcinoms. Scle r: 1 μm. Insets re mgnified imges. 14
Supplementry Tle S1. Clinicl nd pthologicl chrcteristics of ER-negtive rest cncer ptients No. of ptients Age <5 125 5 82 Missing Histologicl Grde 1 14 2 9 3 69 Missing 34 T stge 1 43 2 132 3 3 Missing 2 Node sttus 84 1 6 2 34 3 25 Missing 4 Size <3 mm 14 3 mm 11 Missing 2 PgR Negtive 192 Positive 14 Missing 1 HER2 Negtive 48 Positive 132 Missing 27 15