% ells with ili (mrke y A-Tu) Reltive Luiferse % ells with ili (mrke y Arl13) % ells with ili DOI: 1.138/n2259 A-Tuulin Hoehst % Cilite Non-ilite -Serum 9% 8% 7% 1 6% % 4% +Serum 1 3% 2% 1% % Serum: - + Dnh2+/+ Dnh2-/- Cilite Non-ilite Cilite Non-ilite 8 8 6 6 4 4 2 2 Dnh2: +/+ -/- Dnh2: +/+ -/- NCl LiCl A-Tuulin Hoehst 15μm 7 6 5 4 3 2 1 15μm Averge ili length NCl LiCl e 9 8 7 6 4 3 2 1 LiCl: - - + + Dnh2+/+ Dnh2-/- Figure S1 Cili quntifition in MEFs.. Stining for ili (etylte tuulin, re, rrow) in serum strve MEFs with repplition of serum. These results re quntifie t the right for t lest 2 ells. P<.5, Chi-squre test. Nulei re lele with Hoehst (lue).. Stining for the primry ilium (etylte tuulin, re, rrow) in Dnh2 MEFs revels loss of ili in Dnh2-/- MEFs. These results re quntifie in the pnel on the right for t lest 2 ells. P<.5, Chi-squre test.. Quntifition of perent ilite Dnh2 mutnt MEFs using n ntioy for n lterntive ili mrker Arl13. P<.5, Chi-squre test.. Stining for ili (etylte tuulin, re, rrows) in MEFs serum strve n trete with NCl or LiCl. Averge ili length ws quntifie from over 3 ells from three regions of the well for eh tretment. e. Luiferse tivity in Dnh2 MEFs with tivtion of the pthwy with lithium (LiCl). P<.5, n=3 inepenent experiments, Stuent s t-test. Error rs represent S.E.M. WWW.NATURE.COM/NATURECELLBIOLOGY 1
% ells with ili 75 kd Kif3 sirna 1 2 3 Kif3A α-tuulin WB A-Tuulin Hoehst Cilite Non-ilite % 9% 8% 7% 6% 1 % 4% 3% 2% Kif3 sirna 1 1% % Kif3 sirna β-tenin A-Tuulin β-t Tu Hoehst Untrete ilite nonilite 5μm 5 Figure S2 Kif3 knokown n quntifition of ilition.. Western lot nlysis of whole ell lystes from 293Ts trnsfete with Kif3A sirna. Alph-tuulin is shown s loing ontrol.. Stining for ili (etylte tuulin, re) in MEFs trnsfete with Kif3 sirna #3. Arrows point to ili in ontrol trnsfete ells. Hoehst (lue) lels nulei. Results re quntifie t the right from t lest 2 ells for eh. P<.5, Chi-squre test.. β-tenin-gfp (green) loliztion in untrete ilite (etylte tuulin, re) n nonilite MEFs. Arrow points to sl oy lolize β-tenin in ilite ell. Hoehst (lue) lels nulei. 2 WWW.NATURE.COM/NATURECELLBIOLOGY
Nuler β-tenin (men grey vlue) β-tenin A-Tuulin Hoehst Merge 3 2 1 1 Cilite Non-ilite Cytosol/memrne Nuleus Dnh2: +/+ -/- -/- +/+ Wnt3: - + - + - + - + 1 β-tenin Jn m-414 WB Dnh2: +/+ -/- Tuulin Wnt3: - + - + 1 Jn Tuulin Figure S3 β-tenin n Jn re sequestere in the presene of ili.. Stining for enogenous β-tenin (green) in ilite (tylte tuulin, re) n nonilite (rrow) IMCDs reveling inrese nuler (Hoehst, lue, outline) loliztion of β-tenin in nonilite ells ompre with ilite neighoring ells.. Quntifition of nuler β-tenin stining in ilite n nonilite IMCDs. Men grey vlues were lulte using ImgeJ softwre (NIH) to rete inry msk from Hoehst stining s nuler ounry for mesurement of β-tenin levels. P<.5, n=2 nonilite n n=48 ilite ells, Stuent s t-test. Error rs represent S.E.M.. Western lot (WB) for β-tenin n Jn from nuler preps of Dnh2+/+ n -/- MEFs trete with Wnt3 onitione mei. The inrese in nuler β-tenin levels in Dnh2-/- ells is more pronoune ompre with +/+ ells. Aitionlly, Jn exhiits inrese nuler levels in Dnh2 -/- ells. M414 is shown s nuler loing ontrol n Alph-tuulin is ytosoli ontrol.. Western lot for Jn from totl ell lystes from Dnh2 mutnt n ontrol MEFs trete with Wnt3 onitione or L ell onitione mei. Alph-tuulin is shown s loing ontrol. WWW.NATURE.COM/NATURECELLBIOLOGY 3
Reltive Luiferse -tuulin Jn-GFP Merge A-Tuulin Arl13 5 µm 1 µm 1 µm 15 1 5 β-ctδn: + + + + EV Jn EV Jn Kif3 sirna Wil-type RVxP -Tu Jn-GFP 15 µm 15 µm e 1 kd WBL WCL Sup. Sup. + Txol Pellet Pellet + Txol Jn f Dh2 RNAi: Con sirna 1 sirna 2 sirna 3 DCX 2 Dnh2 α-tuulin terk 2 Dnh1 Figure S4 Loss of ili potentites Jn s role in the Wnt pthwy.. 293T ells stine for ili with ntioy to etylte tuulin (re) isply ler ili with Jn-GFP (green) lolize to the sl oy similr to previously esrie in other ilite ells 2. Hoehst ws use to stin the nuleus (lue).. Cili stining of 293T ells with two inepenent mrkers: etylte tuulin n Arl13. Arl13 revels greter numer of ili (44% of ells) thn etylte tuulin stining (1% of ells).. Luiferse tivity in MEFs trnsfete with Kif3 sirna n overexpresse Jn or EV with tivtion of the pthwy with otrnsfete β-tδn. P<.5, n=3 inepenent experiments, Stuent s t-test. Error rs represent S.E.M.. Loliztion of wil-type n ΔRVxP muttion (GFP tgge onstruts, green) to primry ili (lele y etylte tuulin, re) of MEFs. Hoehst (lue) lels nulei. Arrows point to ili. Arrowhes point to sl oy loliztion of Jn wil-type onstrut. e. Mirotuule ssoition ssy in P5 mouse ereellum lystes followe y western lotting revels enogenous Jn ssoite with repolymerize mirotuules in the Pellet n Pellet + Txol frtions. Blots were proe for Douleortin (DCX) s positive ontrol, n totl ERK s negtive ontrol. α-tuulin is enrihe in pellet frtions. f. Western lot nlysis of whole ell lystes from 3T3s trnsfete with Dnh2 sirna onstruts. Dnh2 levels were speifilly erese in sirna-1 n sirna-2 trnsfete ells without n effet on ynein hevy hin 1 (Dnh1) whih is lso shown s loing ontrol. 4 WWW.NATURE.COM/NATURECELLBIOLOGY
Reltive Luiferse % ells with ili Nonilite MEFs (+ serum) 3 N.S. 2.5 2 1.5 1.5 Dnh2 sirna: - + - + LCM WCM Low ose High ose A-Tuulin Hoehst 6% % 4% 3% 2% 1% % Low ose High ose % ells with 2 ili Aphiiolin.25.2.15.1.5 A-Tuulin Merge Aphiiolin: - + Figure S5 Dnh2 sirna n phiiolin tretment effet on ilition.. Luiferse tivity in MEFs without serum strvtion n trnsfete with 5 pmol Dnh2 sirna n trete with WCM or LCM. N.S.=not signifint, n=3 inepenent experiments, Stuent s t-test. Error rs represent S.E.M.. Stining for ili (etylte tuulin, re, rrow) in MEFs trnsfete with low ose n high ose (3-fol higher) Dnh2 sirna. Hoehst (lue) lels nulei. These results re quntifie in the pnel on the right for t lest 2 ells. P<.5, Chi-squre test.. Stining for ili (etylte tuulin, re, rrows) in serum strve MEFs trete with 1 μg/ ml phiiolin (APH) or vehile.. Quntifition of perent of ells with two ili from t lest 2 ells for eh tretment. P<.5, Chisqure test. WWW.NATURE.COM/NATURECELLBIOLOGY 5
Supplementry Figure 6 Dnh2 mutnt BATgl- Mesnephros BATgl+ E9.5 1 mm 1 mm β-gltosise Hoehst 3 μm 3 μm Lung Dnh2 mutnt Lim u Dnh2 mutnt Dnh2 mutnt Arl13 β-gl Hoehst β-gltosise Arl13 2 μm 2 μm 2 μm 2 μm e β-ctenin Hoehst β-ctenin Arl13 Hoehst Metnephros Dnh2 mutnt 2 μm 2 μm Arl13 Hoehst 2 μm 2 μm 2 μm 2 μm Figure S6 Dnh2 loss les to vrying effets on ili n Wnt signling.. Whole mount imges of X-gl stine E9.5 ontrol n Dnh2 mutnt emryos reveling inrese Wnt tivity in the mi-hinrin region n nsl fols (rrows).. β-gltosise stining (green) of E9.5 BATgl+ n BATgl- ontrol mesonephros (outline). Hoehst (lue) lels nulei.. Wnt reporter stining (β-gltosise, green) in representtive E11.5 lung n lim u of Dnh2 mutnt n ontrol littermte. Arl13 (re) stins ili in these tissues. Cili (rrows) re retine in lung n lim u. Hoehst lels nulei (lue).. High mgnifition imge reveling ili (re, Arl13 stining) in ontrol n Dnh2 mutnt emryos. Hoehst lels nulei (lue). Arrows point to ili, while rrowhe points to pprently ulous ili of Dnh2 mutnt emryo. e. β-tenin stining (re) in E11.5 metnephros (outline) of littermte ontrol n Dnh2 mutnt emryos reveling erese nuler stining (rrows) in mutnts ompre with ontrols. Ajent setions (ottom pnels) were lso stine for ili (Arl13, green). Hoehst stining (lue) lels nulei. 6 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure 5 Jouerin Ki67 α-tuulin 2 2 2 1 1 1 75 75 75 Figure S7 Full western lot sns. WWW.NATURE.COM/NATURECELLBIOLOGY 7