doi:1.138/nture9387 Supplementry Text RANK deletion in tumors nd Cre effects. Southern lotting of the tumors tht developed in RANK mm femles showed deletion of RANK, leit we oserved some residul wild type nd (Supplementry Fig. 5c) tht my e explined y the presence of other cell types nd/or escper cells. We did not oserve differences in tumor onset in Cre-negtive control femles nd littermtes expressing the MMTV-Cre trnsgene indicting tht Cre expression per se does not lter tumor incidence in the MPA/DMBA mmmry tumor model (Supplementry Fig. 5d). Gene expression profiling. Gene expression profiling of mmmry crcinoms from control nd RANK mm femles showed distinctive differences in their moleculr signtures s ssessed y gene clustering (Supplementry Fig. 7). Moreover, using gene set enrichment nlyses (GSEA), genes nnotted to TGFβ signling (TOB1 pthwy), oxidtive phosphoryltion, mitochondril ftty cid β-oxidtion, specific trnscriptionl repressor ctivity, PPRG signling, epithelil to mesenchyml trnsition (EMT), nd MMP ctivity were enriched in tumors from control mice (Supplementry Fig. 7). By contrst, mmmry tumors from RANK mm femles showed enrichment for the p53 hypoxi pthwy, glycolysis, glyconeogenesis, poptosis, polrized growth, nd morphogenesis of n epithelium (Supplementry Fig. 7c). www.nture.com/nature 1
RESEARCH tmrankl srankl Shm MPA 3d 6d 3d 6d 45kD 19kD TM RANKL1 N- 14 44 1 TM RANKL2 N- 1 -C 316 -C 287 -ctin RANKL3 N- 1 -C 199 c reltive mrna expression 16 shm Shm 12 8 4 MPA d Shm MPA e reltive mrna expression 2 16 12 8 4 f Shm shm MPA PRL-R KO Shm MPA MPA srankl -ctin Supplementry Figure 1. The progesterone-derivtive MPA triggers RANKL expression. -d, Induction of RANKL expression y the progesterone-derivtive MPA. Nulliprous 2 WWW.NATURE.COM/NATURE
RESEARCH wild type femles were s.c. implnted with slow-relese MPA pellets or treted with surgery shm., Expression of trnsmemrne (tm) nd solule (s) RANKL protein ws ssyed on isolted mmmry glnd epithelil cells y Western lot 3 nd 6 dys fter MPA pellet implnttion or shm surgery. β-ctin is shown s loding control. Recominnt murine srankl nd recominnt humn tmrankl protein re shown for moleculr size comprison in the right pnels. Dt re representtive of 8 nimls tested., Schemtic representtion of the three RANKL isoforms dpted from Iked et l. 1. The trnsmemrne domin of RANKL1 nd RANKL2 is indicted (TM, lck ox, spnning 48-71). RANKL2 is lcking 14-44 of the intrcellulr domin while RANKL3 is lcking 1-118 spnning the whole intrcellulr domin s well s the trnsmemrne domin. Numers indicte mino cid residues. c, Expression of totl (t) RANKL, RANKL1, RANKL2, nd RANKL3 mrna in purified mmmry epithelil cells fter three dys MPA tretment. β-ctin mrna ws used for normliztion. d, Representtive grose gel of RT-PCR products s in c. e, mrna expression of vrious proteses known to shed RANKL from the surfce of cells. mrna ws determined in purified mmmry epithelil cells y qrt-pcr three dys fter MPA implnttion. β- ctin mrna ws used for normliztion. Dt re shown s fold chnge compred to shm tretment (+/- sem, n=3 per group). f, Induction of solule RANKL protein ssyed on isolted mmmry glnd epithelil cells from control, ut not in prolctin receptor mutnt (PRL-R KO) femles on dy 3 fter s.c. MPA implnttion. Mmmry glnds from shm operted control littermtes nd PRL-R KO femles re shown s controls. P<.5; P <.5; P <.1 (Student s t-test). www.nture.com/nature 3
RESEARCH RANK fl/d, K5-Cre Nulliprous Lcttion L1 fl/+ D c nu/nu Clered wt nu/nu RANK fl/d, K5-Cre nu/nu 5x Supplementry Figure 2. RANK fl/ femles crossed to K5-Cre mice show defective loulo-lveolr development in pregnncy., Whole-mount nlyses of mmmry tissue of nulliprous nd lctting (L1) RANK fl/ 4 WWW.NATURE.COM/NATURE
RESEARCH femles crossed with K5-Cre compred to mmmry glnds of control littermtes. Alveoli in gestting wild-type femles (rrow) form loulo-lveolr structures, wheres this development is rrested t rudimentry lveolr ud (rrow) in K5-Cre RANK fl/ femles., Southern lot of purified mmmry epithelil cells derived from RANK flox/ nd RANK flox/ ; K5-Cre femles. The wild type or floxed RANK llele (fl/+; 9.6 k) nd the deleted RANK llele ( ; 3.9 k) re indicted fter digestion of genomic DNA with PvuII nd SphI. c, Whole-mount nlyses of mmmry glnds from control BALBc nude (nu/nu) femles showing norml loulo-lveolr structures t dy 1 of lcttion (L1), norml loulo-lveolr structures t L1 in clered nu/nu mice trnsplnted with wild type mmmry glnd tissue, nd defective loulo-lveolr development t L1 in clered nu/nu mice trnsplnted with RANK fl/ ; K5-Cre mmmry glnd tissue. Ft pds of nu/nu mice fter surgicl clering re lso shown. All mgnifictions X 5. www.nture.com/nature 5
RESEARCH RANK fl/d, MMTV-Cre Nulliprous Nulliprous RANK fl/d, MMTV-Cre c Lcttion L1 Nulliprous fl/+ D Supplementry Figure 3. Norml formtion of lctting mmmry glnd in pregnnt MMTV-Cre, RANK fl/ femles., H&E nlyses of mmmry tissue of nulliprous littermte control nd RANK fl/ ; MMTV-Cre femles showing norml lveolr/ductl epithelil structures. Mgnifictions X 1 (top) nd X 4 (ottom pnels)., Whole-mount nlyses of mmmry tissue of nulliprous nd lctting (L1) RANK fl/ femles crossed with MMTV-Cre compred to mmmry glnds of control littermtes. MMTV-Cre medited deletion of RANK did not ffect formtion of lctting mmmry glnd. c, Southern lot of purified mmmry epithelil cells derived from RANK flox/ nd RANK flox/ ; MMTV-Cre femles. The wild type or floxed RANK llele (fl/+; 9.6 k) nd the deleted RANK llele ( ; 3.9 k) re indicted fter digestion of genomic DNA with PvuII nd SphI. RANK flox/ ; MMTV-Cre nimls re denoted RANK mm herefter. 6 WWW.NATURE.COM/NATURE
RESEARCH fold chnge Ki67 + cells 35 3 25 2 15 1 5 cini (%) 7 6 5 4 3 2 1 low med high % Ki67 positive cells/cinus c cini (%) 1 8 6 4 2 low med high % Ki67 positive cells/cinus d RANKL PBS e Ki67 positive cells (%) 3 25 2 15 1 5 f Numer of CD24 + CD49 high cells x 1 4 2 15 1 5 PBS RANKL Shm MPA Supplementry Figure 4. MPA nd RANKL induce prolifertion of mmmry epithelil cells. -c, Quntifiction of epithelil prolifertion in mmmry glnds of control littermtes nd RANK mm femles 3 dys fter shm tretment nd MPA implnttion s shown in Fig. 1d., Dt re shown s reltive chnges in totl Ki67 + epithelil cells compred to shm-operted femles of the respective genotype. At lest 1 mmmry glnd epithelil cells were counted per mouse. n = 3 mice per genotype. P <.5; P <.1 (Student s t-test)., Quntifiction of in situ Ki67 immunostined cells per cinus from mmmry glnds of control littermte nd RANK mm femles on dy 3 fter MPA www.nture.com/nature 7
RESEARCH s.c. implnttion. Wheres in control femles ~ 8% of cini showed signs of medium to high prolifertion, more thn 6% of cini in RANK mm femles exhiited very low prolifertion rtes. c, To control for estrus-dependent ckground prolifertion levels, superovulted nd shm operted control littermte nd RANK mm femles were nlysed. At lest 1 cells were counted per mmmry glnd. n = 3 per genotype. In, nd c, dt re shown s percentge of cini/ducts with low (<2% of epithelil cells re Ki67 + ), medium (2-8% of epithelil cells re Ki67 + ) nd high (>8% of epithelil cells re Ki67 + ) numers of proliferting cells (+/- sem). P <.1; P <.3 (Student s t-test). d, Epithelil prolifertion in mmmry glnds of control littermtes nd RANK mm femles 1 dy fter i.p. injection of PBS or RANKL (1µg). Prolifertion ws determined in situ Ki67 immunostining. Mgnifictions X 4. e, Quntifiction of epithelil prolifertion 1 dy fter i.p. injection of PBS or RANKL (1µg). Men percentges of Ki67 positive cells re shown (+/- sem, n=5). P <.3; (Student s t-test). f, Quntifiction of the MSC-enriched CD24 + CD49f high popultion from mmmry glnds of MPA- or shm-treted virgin RANK mm nd littermte control femles. For ll MSCs experiments mice were treted with MPA for 3 dys (+/- sem, n=4 per group). P <.5; P <.1 (Student s t-test). 8 WWW.NATURE.COM/NATURE
RESEARCH Supplementry Figure 5. Delyed onset nd enhnced survivl of RANK mm femle mice in MPA-driven rest cncer.,, MPA induces RANKL expression in mmmry epithelil cells. Nulliprous wild type femles were treted with orl gvge of DMBA or oil vehicle, s.c. implnted with slow- relese MPA pellets, or treted with shm surgery., RANKL mrna ws determined in purified mmmry epithelil cells y qrt-pcr 3 dys fter implnttion/orl gvges. β- ctin mrna ws used for normliztion. Dt re shown s fold chnge compred to shm tretment (+/- sem, n=3 mice per group)., Expression of solule (s) RANKL protein (19kD) ws ssyed on cell lystes from purified mmmry epithelil cells y c fold chnge RANKL mrna 25 2 15 1 5 oil DMBA shm MPA RANK fl/+ RANK fl/d MMTV-Cre+ MMTV-Cre+ fl/+ D d tumor-free mice (%) 1 8 6 4 2 srankl 19kD -ctin Cre + Cre - 1 2 3 4 5 6 time fter lst DMBA (d) e Survivl (%) 1 8 6 4 2 p<.3 5 1 time fter lst DMBA (d) 15 www.nture.com/nature 9
RESEARCH Western lot 3 dys fter tretment with oil vehicle, DMBA, MPA, or shm surgery. β- ctin is shown s loding control. Of note, only MPA ut not DMBA or the oil vehicle lone induced RANKL mrna nd protein expression. c, Representtive Southern lot of MPA/DMBA-induced mmmry tumors derived from RANK flox/+ ; MMTV-Cre nd RANK flox/ ; MMTV-Cre (RANK mm ) femles. The wild type or floxed RANK llele (fl/+; 9.6 k) nd the deleted RANK llele ( ; 3.9 k) re indicted fter digestion of genomic DNA with PvuII nd SphI. All tumors derived from RANK mm femles showed lmost complete deletion. d, Onset of plple mmmry tumors in RANK mm (n=14) nd ge-mtched littermte control femles with MMTV-Cre (Cre+ control; n=13) or without MMTV-Cre (Cre- control; n=9) treted with MPA pellets nd DMBA s indicted in Fig. 2. Dt re shown s percentge of tumor-free mice fter the lst DMBA chllenge. No significnt difference ws found etween the Cre+ nd the Cre- control groups. Medin tumor onset for Cre+ controls ws 9 dys, for Cre- controls 11 dys, nd for RANK mm femles 3 dys fter the lst DMBA tretment. e, Kpln Myer nlysis for overll survivl of RANK mm (n=9) nd ge-mtched littermte control femles (n=9) fter tretment with MPA /DMBA. Medin survivl ws 48 dys for control nd 93 dys fter the lst DMBA tretment for RANK mm femles. 1 WWW.NATURE.COM/NATURE
RESEARCH 7dys fter DMBA E-Cdherin CK14 H&E H&E 22 dys fter DMBA Supplementry Figure 6. Development of squmous denocrcinoms in RANK mm femles.,, Representtive histologicl sections of mmmry tumors isolted from control littermte nd RANK mm femles 7 () nd 22 () dys fter the lst DMBA tretment. H&E nd E-cdherin stinings re shown indicting typicl fetures of ductl denocrcinoms in tumors from control littermtes nd RANK mm femles. Cytokertin 14 (CK14) expression demonstrtes the sl cell origin in oth controls nd RANK mm femles. However, RANK mm femles tend to show chrcteristics of squmous metplsi. All mgnifictions X 2. www.nture.com/nature 11
RESEARCH Gene Sets enriched in control c Gene Sets enriched in TOB1 pthwy TGFB2 TGFB3 CD28 CD3G INFG Oxidtive phosphoryltion NDUFB8 NDUFA5 NDUFA13 NDUFA4 ATP6VB PPA2 CYC1 NDUFA8 NDUFB11 NDUFS6 NDUFS3 ATP5G2 COX6A1 NDUFB5 UQCRQ COX17 NDUFA6 NDUFB3 COX6C ATP6VA2 NDUFV2 COX7A1 NDUFA1 UQCRB COX7B COX8A,5 Mitochondril ftty cid -oxidtion ACADM ACSL3 EHHADH HADHA ACSL1 ACADS Metlloendopeptidse ctivity MMP3 MMP2 ADAMTS2 MMP16 MMP14 ECE2 MEP1A MMP11 MMP15 PITR p53 Hypoxi pthwy AKT1 NFKBIB CDKN1A HIF1A HSPA1A CSNK1A1 Glycolysis & Glyconeogenesis HK1 Apoptosis PG ADH4 ENO1 PKLR LDHC ALDOB BPGM ALDH3A2 ALDH9A1 ALDH3B1 PGA PK LDHA PGK1 PG PFKP TRAF6 CASP3 BCL2L1 TNFRSF1B NFKBIA TNF NTRK1 TFG FAS BOK CASP8 CASP6 MCL1 CASP1 CD4 CASP4 Morphogenesis of n epithelium BCL1 VANGL2 UPK1B UPK3A Trnscriptionl repressor ctivity NCOA1 HDAC5 EID1 HDAC6 PPRG pthwy NCOA1 EP3 LPL CREBBP Site of polrized growth MAPT APBB1 APBB2 PRKCI ANG Epithelil to mesenchyml trnsition TRI8 TGB2 TGFB3 BMP2 HNRPAB CTNNR1 Supplementry Figure 7. Gene expression profiling of RANK mm mmmry cncers. Gene profiling of lte stge tumors from littermte control nd RANK mm femles., Gene clustering from individul tumors isolted from 5 control littermte nd 5 RANK mm femles. The dendrogrm ws generted using hierrchicl clustering. Levels of expression re represented on scle from lue (lowest expression) to red (highest expression).,c, Gene Set Enrichment Anlysis (GSEA) from the sme tumors s shown in, to determine whether defined sets of genes or pthwys show sttisticlly significnt enrichment in the most ltered genes., Gene Sets enriched in mmmry tumors tht developed in control littermte femles. c, Gene Sets enriched in mmmry tumors tht developed in RANK mm femles. GSEA were performed using the GSEA tool t http://www.rod.mit.edu/gse. All dt sets re ville upon requests nd will e pulished online t NCBI. 12 WWW.NATURE.COM/NATURE
RESEARCH c tumor-free mice (%) 1 75 5 25 5 1 15 2 25 3 BAC time fter lst DMBA (dys) thymus hert RANK Mx-Cre Mx-Cre RANK D fl liver spleen MECs fl/+ D spleen hert liver thymus Poly I:C - + - + - + - + BAC fl D d e tumor-free mice (%) (%) 1 75 5 25 25 5 75 1 125 15 175 2 225 time (d) time (dys) NeuT NeuT RANK control fl/+ D NeuT RANK +/D MMTV-Cre+ NeuT RANK fl/d MMTV-Cre+ Supplementry Figure 8. Mmmry cncer onset in Mx-Cre RANK floxed/ nd NeuT RANK mm mice., Onset of plple mmmry tumors in Mx-Cre rnk floxed/ (n=4) nd ge mtched Mx- Cre rnk +/ littermte control femles (n=6) treted with MPA pellets nd DMBA s www.nture.com/nature 13
RESEARCH indicted in Fig. 2. The Mx-driven Cre recominse ws ctivted y four poly I:C injections i.p. over the course of 8 dys (2µg in 2 ml PBS). Dt re shown s percentge of tumor free mice fter the lst DMBA chllenge. No significnt differences were found., Southern lot of the non-deleted RANK floxed llele (fl/+) nd fter induction of deletion ( ) in Mx-Cre rnk floxed/ mice. c, Southern lot of vrious orgns fter induction of deletion ( ) in Mx-Cre rnk floxed/ mice. Note tht while vrious degrees of deletion (5-1%) cn e seen in thymus, hert, liver, nd spleen, deletion of the RANK floxed llele ws not induced in purified mmmry epithelil cells (MECs). d, Onset of plple mmmry tumors in NeuT RANK mm (NeuT, MMTV-Cre rnk floxed/ ) femles (n=18) nd ge mtched NeuT (NeuT MMTV-Cre rnk +/ ) control littermte femles (n=17). No significnt difference ws found. e, Southern lot of tumors derived from NeuT MMTVCre rnk floxed/ nd mmmry cncers from littermte NeuT MMTVCre RANK +/ femles to control for RANK deletion in tumors. 14 WWW.NATURE.COM/NATURE
RESEARCH NeuT+ Cytokertin 5 NeuT+ Cytokertin 14 NeuT- NeuT- c NeuT+ E-cdherin NeuT- d 2 e reltive RANKL mrna expression 15 1 5 RANK RANKL -ctin Supplementry Figure 9. Tumor formtion in NeuT RANK mm femles. -c, Representtive histologicl sections with typicl denocrcinoms detected in control www.nture.com/nature 15
RESEARCH nd NeuT RANK mm femle mice. Cytokertin 5 (), Cytokertin 14 () nd E-cdherin (c) stinings re shown in mmmry glnds of 6-month old femles. Mmmry glnds from littermte RANK mm femles not expressing NeuT (NeuT-) re shown s controls. d, RANKL mrna expression in mmmry epithelil cells (MECs) isolted from either shm- or MPA-treted virgin mice, in MPA-driven mmmry denocrcinoms, nd primry mmmry tumors derived from NeuT, Pym, Neu, nd Neu/TGFβ trnsgenic mice. RANKL mrna ws determined y qrt-pcr. β-ctin mrna ws used for normliztion. Dt re shown s fold chnge compred to shm tretment (+/- sem, n=3 per group). e, RANKL nd RANK protein expression in vrious tumors isolted from the indicted genetic mouse models nd MPA-driven tumors in control RANK /+ nd RANK mm femles. Recominnt murine srankl protein is shown for moleculr size comprison. β-ctin is shown s loding control. 16 WWW.NATURE.COM/NATURE
RESEARCH RANKL RANKL 15 6 12 min P-Akt RANK IKK IkB Akt P-Erk1/2 Id2 Id2 p21 P-NFκB CyclinD1 Erk1/2 P-p38 p38 Supplementry Figure 1. RANKL/RANK downstrem signling in MECs., Schemtic outline of geneticlly confirmed signlling pthwys tht control RANKL/RANK medited formtion of lctting mmmry glnd during pregnncy 2, 3., Western lotting for phosphorylted (P) AKT, totl AKT, phosphorylted (P) ERK1/2, totl ERK1/2, phosphorylted (P) p38-mapk, nd p38-mapk in isolted primry mouse mmmry glnd epithelil cells in response to RANKL stimultion (1µg/ml). Dt re representtive of 4 experiments. www.nture.com/nature 17
RESEARCH Shm P-IkB MPA srankl RANK D/+ Shm MPA Shm MPA CyclinD1 RANKL -ctin Supplementry Figure 11. MPA ctivtes the NFκB pthwy nd triggers CyclinD1 expression vi RANKL/RANK.,, Activtion of the NFκB pthwy nd CyclinD1 expression y MPA. Nulliprous RANK mm nd littermte control femles were s.c. implnted with slow-relese MPA pellets or treted with shm surgery., In situ immunostining to detect phosphorylted (P) IκBα in mmmry epithelil cells of RANK mm nd littermte control femles fter 3d MPA tretment., Western lot nlysis of CyclinD1 nd RANKL in isolted mmmry epithelil cells from RANK mm nd littermte control femles fter 3d MPA tretment. Recominnt, murine srankl protein is shown for moleculr size comprison. β-ctin is shown s loding control. 18 WWW.NATURE.COM/NATURE
RESEARCH RANKL 5 15 3 6 min P-p65 NFkB p65 NFkB P-IkB IkB P-Erk1/2 Erk1/2 P-p38 p38 1x1 5 cells 6 5 4 3 2 1 RANKL 1 2 3 4 time (dys) c d tumor-free mice (%) 1 75 5 25 NFATc1 Dmm 1 2 3 time fter lst DMBA (d) 4 reltive NFATc1 mrna expression 2 1,5 1,5 MECs NFATc1 Dmm MPA tumor Supplementry Figure 12. RANKL/RANK downstrem signling pthwys., Western lotting for phosphorylted (P) p65 NFκB, totl p65 NFκB, phosphorylted (P) IκBα, totl IκBα, phosphorylted (P) ERK1/2, totl ERK1/2, phosphorylted (P) p38-mapk, nd p38-mapk in humn SKBR3 rest cncer cells in response to RANKL stimultion (1µg/ml). Dt re representtive of 3 experiments., Growth curve of SKBR3 rest cncer cells cultured in norml growth medium (control, DMEM supplemented with 1% FCS) or in the presence of RANKL (1µg/ml). Cell numers were determined y counting live cells (trypn lue exclusion) over 3 dys. c, Onset of plple mmmry tumors in NFATc1 mm (n=1) nd ge mtched littermte control (n=16) femles treted with MPA pellets nd DMBA. Dt re shown s percentge of tumor free mice fter the lst DMBA chllenge. No significnt difference ws found. d, Quntifiction of NFATc1 mrna expression in purified mmmry epithelil cells (MECs) nd MPA-driven mmmry tumors from NFATc1 mm nd littermte control femles. mrna ws determined y qrt-pcr. β-ctin mrna ws used for normliztion. Dt re shown s fold chnge compred to control (+/- sem, n=5 per group). P <.5 (Student s t-test). www.nture.com/nature 19
RESEARCH MECs 4 8 12 16 24 hours fter g-irrdition RANKL - + - + - + - + - + - + gh2ax P-Chk1 Chk1 p53 -ctin MECs h 8h MEC 16h 2Gy RANKL.26 24h MECs 24h 2Gry.44 MECs -.36 49 7 41 M4 MECs 11h 2GRy.33 23 3 7 64 13 26 5 61 24 1 54 21 1 M4 M4 M4 g-irrdition counts MECs RANKL.37 48 12 36 MECs 11h 2GRy RANKL.34 SKB3 8h RANKL 1Gry RANKL.25 MECs 24h 2Gry RANKL.46 2 4 6 8 2 1 4 6 8 2 1 4 6 8 2 1 4 6 8 1 19 3 11 64 M4 28 32 7 13 58 2 34 33 1 M4 M4 M4 g-irrdition + RANKL c 2 4 6 8 2 1 4 6 8 2 1 4 6 8 2 1 4 6 8 1 SKBR3 h 8h 24h 48h SKB3 24h -.31 5 55 7 24 29 1 64 M4 5 44 24 19 5 48 28 13 3 g-irrdition counts SKB3 24h R.32 SKB3 8h RANKL 2Gry RANKL.21 2 4 6 8 2 14 6 2 4 6 2 4 6 59 F L 2 - A 25 29 1 6 64 6 14 34 35 8 11 55 23 4 M4 M4 6 g-irrdition + RANKL 2 4 6 8 21 4 6 8 2 1 4 6 2 4 6 Suppl. Figure S13 Supplementry Figure 13. RANKL protects primry murine mmmry epithelil cells nd humn SKBR3 rest cncer cells from poptosis in response to γ-irrdition., Western Blot nlysis for γh2ax, phosphorylted (P) Chk1, totl Chk1, p53 nd β- ctin in isolted primry mouse mmmry glnd epithelil cells in response to γ- irrdition (2 Gry) in the presence (1µg/ml) or sence of RANKL stimultion.,c, FACS nlysis of propidium iodide (PI) stined, mmmry epithelil cells nd c, SKBR3 humn rest cncer cells fter γ-irrdition (2 Gry) in the sence or presence (1µg/ml) of RANKL. Dt re representtive of t lest 3 experiments. Apoptotic cells with DNA content < 2n pper in the su-g1 region. Percent of cells in su-g1 (), G1-phse (), S/G2/M-phse () s well s polyploid cells with DNA content > 4n re given for the indicted time points. 2 WWW.NATURE.COM/NATURE
SKB3 24h D.27 SKB3 36h DR test next dy.37 2 4 6 8 1 2 4 6 8 1 M4 SKB3 36h D test next dy.36 M4 M4 2 4 6 8 1 RESEARCH MECs 24h 36h 11 4 45 47 3 14 47 37 3 M 5 SKBR3 24h SKB3 24h -.25 36h M4 5 52 26 17 18 41 23 17 MECs 24h untreted.15 MECs 36h R.18 SKB3 24h R.26 SKB3 24h R.5 RANKL 2 4 6 8 3 6 45 46 2 9 48 42 2 M 5 2 4 6 8 RANKL 2 4 6 8 2 14 6 8 5 57 27 12 7 46 27 19 M4 M4 Dox counts MECs 24h D/R.18 MECs 36h D.19 2 4 6 8 2 4 6 8 1 14 F L 2 - A 73 3 84 8 7 1 75 M Dox counts 2 4 6 8 2 14 6 8 1 1 3 23 71 16 49 2 3 Dox + RANKL MECs 24h D R nchhltig.19 2 4 6 8 F L 2 - A MECs 36h D/R.2 2 4 6 8 1 15 72 3 45 22 23 1 M Dox + RANKL 11 SKB3 24h DR.28 8 68 12 35 16 4 7 M4 2 4 6 8 2 4 6 8 2 4 6 8 1 c MECs fold chnge RANKL mrna 18 16 14 12 1 8 6 4 2 + +RANKL 6h g-irrdition fter irrdition 6h g-irrdition fter irrdition +RANKL + RANKL Bim Pum Nox Supplementry Figure 14. RANKL protects primry murine mmmry epithelil cells nd humn SKBR3 rest cncer cells from poptosis in response to doxoruicin.,, FACS nlysis of, mmmry epithelil cells nd, SKBR3 humn rest cncer cells incuted with the genotoxic gent doxoruicin (Dox, 1µΜ) in the presence (1µg/ml) or sence of RANKL. Dt re representtive of t lest 3 experiments. Percent of cells in su-g1 (), G1-phse (), S/G2/M-phse () s well s polyploid cells with DNA content >4n re given for 24 nd 36 hours fter doxoruicin tretment. c, mrna expression of pro-poptotic genes Bim, Pum, nd Nox 6 hours fter γ-irrdition (2 Gry) in the presence (1µg/ml) or sence of RANKL stimultion. β-ctin mrna ws used for normliztion. Dt re shown s fold chnge compred to control (+/- sem, n=3 per group). P <.5; P <.5 (Student s t-test). www.nture.com/nature 21
RESEARCH IKK Dmm 5Gy + MPA 5Gy % poptosis 2 1,5 1,5 shm MPA 5Gy 5Gy+MPA IKK Dmm Supplementry Figure 15. IKKα medites MPA-induced protection from rdition-induced epithelil poptosis.,, Reduced induction of mmmry epithelil cell poptosis in response to γ-irrdition in IKKα mm femles. Littermtes control nd IKKα mm femles were either shm operted or implnted with n MPA pellet nd γ-irrdited (5 Gry). MPA pellets were implnted 3 dys efore γ-irrdition. 24 hours fter irrdition, poptosis ws detected y immunostining for ctive Cspse 3., Apoptotic nuclei of epithelil cells (rrows) re shown for representtive mmmry glnd sections. Mgnifictions X 4., Quntifiction of mmmry epithelil poptosis. A minimum of 5 nuclei ws counted for ech mouse. Results shown re men vlues (+/- sem, n = 3 mice per group). P <.5 (Student s t-test). 22 WWW.NATURE.COM/NATURE
RESEARCH Densitometry Suppl. Fig. 1 Suppl. Fig. 1f fold increse srankl 16 12 8 4 3d 6d 3d 6d Shm MPA 16 12 fold increse srankl 8 4 Contorl PRL-R KO Contorl PRL-R KO Shm MPA Fig. 3 Fig. 3 fold increse P-IKK 4 2 15 6 12 fold increse IKK 2 1 RANK KO IKK KO fold increse P-p65 8 6 4 2 15 6 12 fold increse P-p65 2 1 RANK KO IKK KO fold increse P-IkB 1 8 6 4 2 15 6 12 fold increse IkB 6 4 2 RANK KO IKK KO time (min) Supplementry Figure 16. Quntifiction of Western lots. Densitometry ws performed on t lest three independent Western lots per experiment. Dt re shown for Western lots in Supplementry Fig. 1, Supplementry Fig. 1f, Fig. 3, nd Fig. 3. Expression vlues for the indicted proteins were normlized to β-ctin loding controls. For quntifiction of phosphoryltion dt were normlized to the respective totl protein nds. P <.5; P <.1 (Student s t-test). www.nture.com/nature 23
RESEARCH Supplementl References 1. Iked, T., Ksi, M., Utsuym, M. & Hirokw, K. Determintion of three isoforms of the receptor ctivtor of nucler fctor-kppb lignd nd their differentil expression in one nd thymus. Endocrinology 142, 1419-26 (21). 2. Co, Y. et l. IKKlph provides n essentil link etween RANK signling nd cyclin D1 expression during mmmry glnd development. Cell 17, 763-75 (21). 3. Kim, N.S. et l. Receptor ctivtor of NF-kppB lignd regultes the prolifertion of mmmry epithelil cells vi Id2. Mol Cell Biol 26, 12-13 (26). 24 WWW.NATURE.COM/NATURE