Alimonti_Supplementry Figure 1 hy 3 4 5 3 Neo 4 5 5 Proe 5 Proe hy/ hy/ /- - 3 6 Neo β-tin d Reltive Protein level (% ) 15 1 5 hy/ /- Reltive Gene Expr. (% ) 15 1 5 hy/ /- Supplementry Figure 1 Chrteriztion of hy/ mouse emryoni firolsts (MEFs). () Shemti representtion the genomi onstrut of the hypomorphi (hy) llele hroring the neomyin ssette, nd the wild-type (), nd null ( ) lleles. The genomi sequene is depited s white line, with gry oxes representing exons 3, 4, 5 nd 6. The retngulr ox represent the Neo resistne the gry tringles represent the loxp sites. The genomi frgment (proe 6.1), used s proe for Southern lot nlysis is shown s red r. () Western lot nlysis of, hy/ nd /- MEFs showing sutle redution of protein levels in hy/ MEFs when ompred with the nd /- ells. Arevition ode used is: hy/ = mie hving hypomorphi nd llele; /- = mie hving nd null llele () Quntifition of protein level from, hy/ nd /- littermte MEFs. Error rs show S.D. from three independent experiments. (d) Quntifition of mrna levels in, hy/ nd /- MEFs. Error rs show S.D. from three independent experiments. Nture Genetis: doi:1.138/ng.556
Alimonti _Supplementry Figure S2 (n=3) hy/ (n=32) / (n=24) 1 8 6 4 2 Lymph-node Averge dimeter (inh) Perentge of mie ffeted with Lymphodenopthy 12.9.8 n=7.7.6 1 m.5.4.3.2 n=4 hy/ /-.1 1-3 4-6 7-9 1-12 13-15 16-18 19-21 >22 p=.2 Time (months) p=.8 d Spleen.35 Averge weigth(gr) hy/ /- n=5 n=4.3 n=5.25.2 hy/ /- 1 m n=3 Spleen Lymph-node hy/ 4X.15 4X.1 /-.5 hy/ /- 4X p=.4 p=.1 4X hy/ e liver 4X kidney 4X Supplementry Figure 2 Averge size of lymph nodes nd spleen in, hy/ nd /- mie. () Lymphodenopthy disese-free survivl in the, hy/ nd /- popultion; n= numer of nimls nlyzed. (-) Anlysis nd representtive imges of the verge size of the lymph nodes () nd spleen () of the, hy/ nd /- t the time of detetion of the utoimmune disorder. n= numer of nimls nlyzed. p= sttistil signifine. Error rs show S.D. (d) Hemtoxylin nd eosin (H&E) nlysis of spleens nd lymph-nodes showing lymphosplenomegly in hy/ nd /- mie. Inset: H&E stining of spleen nd lymph-node from wild-type mouse, showing spleen with well-defined white nd red pulp nd lymph-node with well-delineted mirontomil fetures. Note the hypertive white pulp nd the prtil effement of the spleen histology of the mutnt mie. Note lso the hyper retive germinl enters with the prtil effement of the norml ortiomedullry oundries of the hy/ nd /- lymph nodes. (e) H&E nlysis shows inflmmtory infiltrtions in the liver nd kidney of the hy/ mie. Nture Genetis: doi:1.138/ng.556
Alimonti_Supplementry Figure 3 hy/ /- Uterine dysplsi 2X 2X Lung Cner Intestinl Polyp hy/ 2X 2X Supplementry Figure 3 hy/ mie develop tumours of different histology fter long lteny. () Representtive H&E imges of hy/ nd /- uterus showing signs of uterine dysplsi. () (Left imge) H&E stining of multi-fol lung denorinom in hy/ femle mouse. (Right imge) Intestinl polyp from 16 month old hy/ mle (see lso Tle 1). Nture Genetis: doi:1.138/ng.556
Alimonti_Supplementry Figure 4 MAMMARY GLANDS lox PCR UTERUS PCR MAMMARY GLANDS UTERUS d 7k MAMMARY TUMORS e IHC in mmmry glnds 5k 7k 262 266 267 /- Uterus 144 yy8 19 hy/- 4X 4X hy/ 5k 244 232 251 253 277 /- 4X /- 4X hy/- 7k Uterus 5k 12 yy4 yy7 hy/- Supplementry Figure 4 hypomorphi mie develop mmmry nd uterine tumours in sene of LOH. () Sheme illustrting the PCR strtegy nd the PCR primers dopted for the LOH nlysis in the hy/ mie. () PCR nlysis of norml tissue, mmmry nd uterine tumours from, hy/ nd /- mie using the lox () nd () primers. Note tht three tumours for eh genotype were nlyzed for the LOH nlysis. (d) Southern lot nlysis of norml tissue, mmmry nd uterine tumours from, /- nd hy/- mie showing the sene of LOH in these tumours. Four tumours for eh genotype were nlyzed. (e) Representtive imges of immunohistohemistry nlysis for in the sme tumours s in (). Note tht tissues from oth hy/ nd /- mmmry epithelium show immunoretivity for. Nture Genetis: doi:1.138/ng.556
Alimonti_Supplementry Figure 5 hy/ Mmmry tissue H&E 1 1X 1X 1X β-tin Reltive Protein level (% ) 1X pakt 1X.5 / hy/ /- / hy/ /- 15 1 5 1X.8 Supplementry Figure 5 Anlysis of pre-neoplsti mmmry glnds in ontrol nd hy/mie. () H&E nd immunohistohemistry nlysis for nd pakt in 2 months old nd hy/ virgin littermte femle mie. () (Upper pnel) Western lot nlysis for nd quntifition of protein level (Lower pnel) in two month old ontrol nd hy/ mmmry glnds showing the presene of protein levels in ll the tissues nlyzed. Error rs show S.D. from three independent experiments. Nture Genetis: doi:1.138/ng.556
Alimonti _Supplementry Figure 6 - - - d3 d2 sicontrol si Trnsripts (Reltive to Hprt) 12 1 8 6 4 2 1.81.62 β-tin SiControl Si (d3) Reltive ell numer (OD 595 nm) 1,6 1,4 1,2 1,8,6,4,2 48 h p=.3 SiControl Si (d3) Supplementry Figure 6 A sutle down regultion of level y sirna promotes hyper-prolifertion in MMECs derived from mie of C57/BL6 pure geneti kground. (Left pnel) Down regultion of protein nd gene expression levels y seril dilutions of sirna in MMECs. (Right pnel) Prolifertion of MMECs derived from C57/BL6 mie, fter trnsfetion with the sirna dilution numer 3 (d3) in MMECs. Error rs show S.D. from three independent experiments. p= sttistil signifine. Nture Genetis: doi:1.138/ng.556
Alimonti _Supplementry Figure 7 1.5 rest tumors norml rest GNF2_CDC2 GNF2_CSK1B Brest tumors PTEN Brest tumors Norml Brest Norml Brest 1,32 PTEN expression in rest tumors reltive to norml smples -1.5 Controls High PTEN expression,99,66,33 GSM85485 GSM85493 GSM85482 GSM85481 GSM8559 GSM85479 GSM85491 GSM85494 GSM85483 GSM85489 GSM85475 GSM85478 GSM85473 GSM85474 GSM85496 GSM85512 GSM8551 GSM85511 GSM8552 GSM8554 GSM85487 GSM85484 GSM8557 GSM85476 GSM8555 GSM85492 GSM85499 GSM8549 GSM85486 GSM85488 GSM8551 GSM8558 GSM855 GSM85497 GSM8548 GSM8556 GSM85477 GSM85498 GSM85495 GSM8553 GSM85514 GSM85515 GSM85513 GSM85517 GSM85516 GSM85519 GSM85518-4 4 Ptients GNF2_CDC2 Brest tumors Norml Brest -4 4 PTEN gene expression (%) > 65 < 65-4 Numer of ptients Perentge of ptients 8 2 32 8 4 GSM85498 (PTEN 7%) ID GSM85485 GSM85482 GSM855 GSM8548 GSM85498 GSM85495 PTEN mrna 38 54 1496 1529 1588 1658 Reltive PTEN expression 17 22 67 67 7 73 GSM85495 (PTEN 73%) PTEN IHC # * 4X # 2X GSM85482 (PTEN 22%) GSM85485 (PTEN 17%) # * 4X *Strom 4X # Norml epithelium Supplementry Figure 7 Chrteriztion of humn rest ner smples with deresed PTEN expression. () Gene expression profile from ontrol nd rest humn tumour smples with low/sent PTEN expression (upper pnel). Note tht these tumours show the sme up-regultion of gene sets enrihed in the hy/ MEFs. See lso Supplementry Fig. 8 for the GSEA nlysis of the enrihed gene sets in the ptients nlyzed nd http://www.rod.mit.edu/gse/msigd/genesets. jsp?olletion=cgn for the orresponding gene set pge. () Histogrm showing the distriution of PTEN mrna expression in norml mmmry tissue (lk rs) nd tumour speimens with sutle (PTEN mrna> 65%, ornge rs) nd pronouned (PTEN mrna <65%, lue rs) redution in PTEN mrna expression. The tle shows the numer nd perentge of ptient tumour smples exhiiting sutle (>65%) or more drmti (<65%) redution in PTEN mrna expression ompred to the verge PTEN expression in the norml mmmry tissue smples (n=7). () (Left pnel) Sore of PTEN immunoretivity in suset of ptients from () with vrying levels of PTEN mrna (showed s solute signl intensity vlues nd reltive expression ompred to norml mmmry tissue smples). (Right pnels) Representtive immunohistohemistry nlysis for PTEN in iopsies showing either sutle or profound dereses in PTEN mrna (orresponding PTEN mrna level reltive to norml mmmry tissue is indited in rkets) Nture Genetis: doi:1.138/ng.556
Alimonti_Supplementry Figure 8-4.. rest tumor d 4. norml rest e f g Supplementry Figure 8. Gene expression profile in ontrol nd ptients ffeted y invsive rest ners. (Upper pnels) Enrihment plot ssessed y GSEA for GNF2_BUB1(), GNF2_CDC2 (), GNF2_CDC2 (), GNF2_MCM4 (d), GNF2_CENPE (e), GNF2_CKS2 (f) nd GNF2_CKSIB (g) gene sets in tumours smples nd in the hy/ MEFs. Genes in the linege-speifi gene sets re mrked with vertil rs, nd the enrihment sore is shown in green. (Lower pnels) Mrker genes of the GNF2_BUB1(), GNF2_CDC2 (), GNF2_CDC2 (), GNF2_ MCM4 (d), GNF2_CENPE (e), GNF2_CKS2(f) nd GNF2_CKSIB (g) gene sets, in ptients ffeted y rest ner (with deresed gene expression for PTEN) ompred to ontrols, rnked y signl to noise rtio Nture Genetis: doi:1.138/ng.556
Supplementry Tle 1. Primer sequenes used for genotyping, exons muttionl nlysis nd sirna experiment. GENOTYPING Forwrd 5 >3 Reverse 5 >3 TGGGAAGAACCTAGCTTGGAGG TTCCATTTGTCACGTCCTGCAC ACTCTACCAGCCCAAGGCCCGG LPTEN1 TGTTTTTGACCAATTAAAGTAGGCTGTG AAAAGTTCCCCTGCTGATGATTTGT MUTATIONAL ANALYSIS exon Forwrd 5 >3 Reverse 5 >3 1 AGTCCAGAGCCATTTCCATC TCTAGAAATGCGCCCAGAAT 2 CTAGCGTGGGAAAAGCTAA CCAAAACACATCAAAATGCAA 3 CTGTTTTAGTCCTGTGCAGC CAGGAATCCAGTTCCTCAAA 4 AGCGCAGTGTTTTTACATGA TCACCAGGCAGTAAAAGAC 5 TGGAGTGAAGAGCACAATGC CACAAAGAGGGAGGAAGGAA 6 TTTGTCTCCCTCCTCCCTCT CACCAAAATTCATTGGGTTAGC 7 GAAGTCCTTACATGGGTTGG AAGGCTTTAAGCAAAAGGTCTG 8 CACAAGGTGTTTGCCTTCAC CAGAAAAGGAAAGGCTACGC 9 AAAAGCAGTGCCCTTCAGA CCTATAACTGCAATCTGACA sirna EXPERIMENT sirna ControlsiRNA AGACCAUAACCCACCACAG AAGGAGACGAGCAAGAGAA Nture Genetis: doi:1.138/ng.556