DOI:.3/n95 Thymus Kiney (kd) TA T7 T TA T7 T Hert TA T7 T: +Dox Cylin B (kd) Thymus Kiney Hert TA T5 T TA T5 T TA T5 T: +Dox Cylin B Poneu S Poneu S CnB T7 CnB T Thymus (kd) + Liver Colon + + (kd) Thymus + Liver Colon + + : Dox Poneu S Supplementry Figure Anlysis of CnB n CnB trnsgene expression. ( n ) Western lot nlysis of tissue extrts from -week-ol ox-trete HA-CnB (strins T7 n T), HA-CnB (strins T5 n T) n ontrol (TA) trnsgeni mie. Poneu S stining of lotte proteins serve s loing ontrol () Immunolots of tissue extrts from -week-ol ox-trete n untrete HA-CnB T7 or HA-CnB T trnsgeni mie emonstrting tht trnsgene expression is tightly ontrolle y ox. All lots re representtive of two inepenent experiments. WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.
G G Pro Promet Met An G G Pro Promet Met An G Cylin B ph3 G P PM M A Cylin B integrte ensity (.u.) x 5 5 G G P PM M A Cylin B ph3 Little higher G ps-cdks γ-tuulin G P PM M A p-ser CDKs sustrte integrte ensity(.u.) x G G P PM M A ps-cdks γ-tuulin Supplementry Figure Cylin B protein levels n Ck sustrte phosphoryltion re inrese in CnB trnsgeni MEFs uring mitosis. () Dox-trete n untrete CnB T7 MEFs t vrious stges of ell yle progression stine for ylin B. P-H3 S stining ws use for ell yle stging. () Quntifition of ylin B signls of ox-trete n untrete CnB T7 MEFs t vrious stges of the ell yle. () As in ut stine for phosphorylte Ck sustrtes. g-tuulin stining ws use for ell yle stging. () Quntifition of pck sustrte signls of ox-trete n untrete CnB T7 MEFs t vrious stges of the ell yle. ws visulize with Hoehst. Sle rs, µm. Dt in n represent men ± s.e.m. (per group, we nlyze ells in, n ells in ). Sttistil signifine ws etermine y two-tile, unpire t-test. Sttistis soure t re provie in Supplementry Tle. p <.5, p <., p <.. WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.
G G Pro Promet Met An G G Pro Promet Met An G Cylin B γ-tuulin G P PM M A Cylin B integrte ensity (.u.) x G G P PM M A Cylin B γ-tuulin G ps-cdks γ-tuulin G P PM M A p-ser CDKs sustrte integrte ensity (.u.) x G G P PM M A ps-cdks γ-tuulin Supplementry Figure 3 Cylin B protein levels n Ck sustrte phosphoryltion re inrese in CnB trnsgeni MEFs throughout the ell yle. () Dox-trete n untrete CnB T MEFs t vrious stges of ell yle progression stine for ylin B. g-tuulin stining ws use for ell yle stging. () Quntifition of ylin B signls of ox-trete n untrete CnB T MEFs t vrious stges of the ell yle. () As in ut stine for phosphorylte Ck sustrtes. g-tuulin stining ws use for ell yle stging. () Quntifition of pck sustrte signls of ox-trete n untrete CnB T MEFs t vrious stges of the ell yle. ws visulize with Hoehst. Sle rs, µm. Dt in n represent men ± s.e.m. (per group, we nlyze ells in n ). Sttistil signifine ws etermine y two-tile, unpire t-test. Sttistis soure t re provie in Supplementry Tle. p <.5, p <., p <.. WWW.NATURE.COM/NATURECELLBIOLOGY 3 Mmilln Pulishers Limite. All rights reserve.
CB CB P-PM PM-M M-A P-A PM-A Sup Fig. Nm n vn Deursen Cylin B fluoresene in nphse (.u. x ) Norml (n=7) + Briges (n=) Kinetis of levge (%) 9 7 3 - -9 - -3 Dox (norml; n=3) +Dox (norml; n=) +Dox (riges; n=7) +Dox (lgging; n=) Anphse onset Time (min) 9 3 (kd) Dox mcherry EGFP Merge mcherry EGFP Merge CnB T7 MEFs +Dox -HA Atin e Cy B Top Centromeres Top Merge + f i % ells with RPA foi 3 + + + +: Dox + + + +: APH CnB T7 CnB T 3 Durtion of S/G (min) j g A numer of ells (X ) % of ells with oth HAx n 53BP foi h CnB + CnB +: Dox CnB T7 CnB T Time (min) Dy Dy Dy Dy3 3 Dy Dy5 5 Dys 35 3 5 5 P PM M P-A PM-A 3 5 Dys Supplementry Figure Cylin B overexpression oes not pper to isrupt erly mitosis. () Quntifition of totl ylin B signl in ox-trete CnB T7 nphses with n without hromtin riges. Dt represent men ± s.e.m. Three inepenent MEF lines were inlue in the nlysis. () Time-lpse imges of iosensor-positive CnB T MEFs progressing through mitosis in the sene or presene of trnsgeni ylin B. Brs, 5 mm. () Quntifition of iosensor fluoresene t hromosomes s ells progress through mitosis. Dt represent men ± s.e.m. from five inepenent experiments. () Western lot nlysis of lystes from inue n noninue CnB T7 MEFs for Top (lots re representtive of two inepenent experiments). (e) Imges of CnB T7 prometphses immunostine for entromeres n Top. Insets show enlrge entromeres from oxe region. Br, 5 mm. (f n g) Quntittion of RPA- or ghax/53bp-positive foi in inue n non-inue CnB T7 n CnB T MEFs. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). (h) Timing of erly stges of mitosis of inue n non-inue CnB T7 MEFs y live-ell imging. P, prophse; PM, prometphse; M, metphse; n A, nphse onset. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line) (i) Anlysis of S/G urtion in inue n non-inue CnB T7 MEFs using FUCCI tehnology. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). (j) Growth urves of inue n non-inue CnB T7 n CnB T MEFs (P3). Dt represent men ± s.e.m. from three inepenent MEF lines. p <.5 enotes sttilly signifint ell numer t y 5. Sttistil signifine ws etermine y two-tile unpire t-test. Sttistis soure t re provie in Supplementry Tle. WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.
CnBT7 CnBT CnBT7 CnBT Durtion of mitoti rrest in txol (h) Pro Promet Met An CnB T7 CnB T Dox 3 Mitoti rrest in noozole (h) +Dox 3 Mitoti rrest in txol (h) Alignment efet (%) + :Dox CnB T Normlize fluores. intensity 3 9 Time (se) j g Dox +Dox CnB T Cylin B Centrin Merge + Prophse ells with premture entrosome seprtion (%) Metphses with norml spinle geometry (%) 7 3 7 3.5 5 BI3 (nm) 5 nm 5 nm nm nm MLN5 (nm) e S/G Mitosis h CnB T + Dox HA Centrin Merge + p-bur T intensity (.u. X 3 ) p-bur T p-plk T Centromeres f intensity (.u. X 3 ) i k l m Dox +Dox p-auror A intensity t entrosomes (.u.) X 3 3 P PM M A p-plk T intensity t entrosomes (.u.) X 3 + Dox CnB T Centrosome istne (µm) MLN5 (nm) Spinle pole-to-ortex istne (Rtio).... + Dox CnB T Supplementry Figure 5 Chrteriztion of the hromosome lgging phenotype of ylin B overexpressing ells. () Noozole- n txolhllenge ssys on CnB T7 n CnB T MEFs with or without trnsgene inution. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). () Quntifition of ells with misligne hromosomes following relese from monstrol n MG3 exposure. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). () Mesurement of kinetohore-mirotuule ynmis in ylin B trnsgeni MEFs. Normlize fluoresene intensity over time fter phototivtion of spinles in prometphse ells. Dt represent men± s.e.m. of ells per genotype. ( n e) Imges of methnol-fixe CnB T MEFs immunostine for the entrosome mrker Centrin n either ylin B () or HA (e). ws visulize with Hoehst. Sle r, µm. (f) Dox-trete CnB T MEFs in G nlyze for entrosome istne n level of HA ylin B expression (9 ells were nlyze). Note tht there is orreltion etween level of overexpression n entrosome istne. (g) Aelerte entrosome seprtion t vrious onentrtions of Plk inhiitor. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). Cells with entrosome istnes mm were lssifie s elerte. (h) Quntittion of p-auror-a signls t entrosomes t the vrious stges of mitosis. Dt represent men ± s.e.m. of 7- ells per group. (i) Quntittion of p-plk T signl t entrosomes in G CnB T MEFs (.u., ritrry unit). Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). (j) Iniene of spinle geometry normlities t vrious onentrtions of Auror A inhiitor MLN5. Dt represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). (k) Imges of CnB T prometphses immunostine for p-plk T, p-bur T, entromeres. p-bur T phosphoryltion y p-plk T meites reruitment of PPA to kinetohores to prevent unontrolle Auror B tivity n estiliztion of mirotuulekinetohore tthments. ws visulize with Hoehst. Br, µm. (l) Quntifition of p-bur T signl in presene or sene of elevte ylin B. Note tht prometphses umulte norml levels of p-bur T, initing tht tthment error orretion t kinetohores is unperture when ylin B levels re upregulte. Dt represent men of ells per group. (m) Spinle position mesurements. Dt represent men ± s.. of ells per group. Sttistil signifine for ws etermine y two-tile, unpire t-test. Sttistis soure t re provie in Supplementry Tle. WWW.NATURE.COM/NATURECELLBIOLOGY 5 Mmilln Pulishers Limite. All rights reserve.
(kd) WT p53 / Auror A Plk Atin Poneu S Plk- reltive fol expression 5 3 Primer set WT MEFs p53 / MEFs Primer set Auror-A reltive fol expression Primer set Primer set p-auror intensity t entrosomes (.u.) X 3 WT MEFs + ontrol shrna p53 / MEFs + ontrol shrna p53 / MEFs + mcb shrna ns 7 5 3 p-plk intensity t entrosomes (.u.) X 3 ns Supplementry Figure p53 loss inreses Auror A n Plk trnsript n protein levels. () Western lots of extrts from wiltype n p53 / MEFs. Atin proe n Poneu S stining serve s loing ontrols (lots re representtive of two inepenent experiments). () Aurk n Plk trnsript levels in wiltype n p53 / MEFs nlyze y qrt-pcr. Dt represent men ± s.e.m. from three inepenent experiments (we mesure uplite of two inepenent MEF lines per experiment). GAPDH trnsript levels were use for normliztion. Vlues were normlize ginst wiltype vlues. Consistent with eviene tht p53 meites trnsriptionl repression of Plk, we fin tht Plk is upregulte in p53 / MEFs. However, it is importnt to emphsize tht even though Plk expression is upregulte, Plk hypertivity in p53 / MEFs remins fully epenent on Auror A hypertivity s low mounts of Auror A kinse MLN5 inhiitor restore proper timing of entrosome seprtion, spinle geometry, n urte hromosome segregtion (see Fig. 5g-i). () Quntittion of p-auror A intensity t entrosomes in G ells. () Quntittion of p-plk T intensity t entrosomes G ells. Dt in n represent men ± s.e.m. from three inepenent MEF lines (we nlyze ells per line). Sttistil signifine ws etermine y two-tile, unpire t-test. Sttistis soure t re provie in Supplementry Tle. p <.5, p <.. WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.
Norml lung Aenoms Squmous ell Norml lung Aenoms Squmous ell Norml lung Aenoms Squmous ell rinoms rinoms rinoms CCNB reltive fol expression 3 CCNB reltive fol expression BUB reltive fol expression Ki-7 reltive fol expression - Norml lung Lung enoms Squmous ell rinoms Supplementry Figure 7 CCNB n CCNB re frequently overexpresse in humn lung rinoms. CCNB (), CCNB (), Ki7 () n BUB () trnsript levels in norml humn lung tissue, lung enorinoms, n squmous ell lung rinoms nlyze y qrt-pcr. Dt represent the men of uplite mesurements. TBP trnsript levels were use for normliztion. The reltive fol CCNB, CCNB, Ki7 or BUB trnsript in tumors ws normlize to the verge of the norml lung tissue vlues. Ki7 n BUB trnsripts were use s prolifertion n mitoti mrkers, respetively. WWW.NATURE.COM/NATURECELLBIOLOGY 7 Mmilln Pulishers Limite. All rights reserve.
Tumors/mouse (smll intestine) 7 3 TA;Ap +/min CnB T7 ;Ap +/min CnB T ;Ap +/min n= n=3 n= TA;APC +/min CnB T7 ;APC +/min CnB T ;APC +/min Supplementry Figure Quntittion of smll intestinl tumors n olon tumor histopthology 3 y fter ox withrwl. () Tumor multipliity in smll intestine. n represents the numer of mie nlyze. Dt represent men ± s.e.m. Sttistil signifine ws etermine y two-tile unpire t-test. () Hemtoxylin-eosin-stine setions of oloni polyps of mie tht were tken off ox for the lst 3 ys of the experiment. Blk n re sle rs re mm n µm, respetively. WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.
Fig. +Dox Fig. CnB T7 MEFs +Dox Dox % % % % % Lyste % : loe KH HYG R ells lones HA-ylin B α-cylin B HA-ylin B Fig. CnB T7 CnB T Dox +Dox Dox +Dox α-atin HA-ylin B HA-ylin B Fig. e CnB T MEFs +Dox Dox % % % % % Lyste % : loe α-cylin B Cylin B α-cylin B Atin α-atin Supplementry Figure 9 The unroppe films showing the key experiments isplye in min figures. Dshe oxes mrk regions tht were roppe. WWW.NATURE.COM/NATURECELLBIOLOGY 9 Mmilln Pulishers Limite. All rights reserve.
Fig. f Spleen TA T7 T Lung TA T7 T Colon TA T7 T Liver TA T7 T Fig. 3g CnB T + : Dox p-t plk p-auror A α-ylin B Cylin B Atin Poneu S Fig. g Spleen Lung Colon TA T5 T TA T5 T TA T5 T TA T5 T Liver α-ylin B Poneu S Fig. Control mcb shrna mcb shrna Cylin B Cylin B Fig. j Control hcb shrna hcb shrna Cylin B Cylin B Atin Atin Supplementry Figure 9 ontinue The unroppe films showing the key experiments isplye in min figures. Dshe oxes mrk regions tht were roppe. WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.
Supplementry Tle Spontneous tumors of CnB n CnB trnsgeni mie re neuploi. Interphse FISH for hromosomes n 7 on tumor ells from CnB or CnB trnsgeni mie. Corresponing tissues from ge-mthe ontrol (TA) mie were use s ontrols. Supplementry Tle Sttistis soure t WWW.NATURE.COM/NATURECELLBIOLOGY Mmilln Pulishers Limite. All rights reserve.