SUPPLEMENTARY INFORMATION

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1 SUPPEMENTARY INFORMATION DOI: 1.138/ncb956 Norml CIS Invsive crcinom 4 months months b Bldder #1 Bldder # Bldder #3 6 months (Invsive crcinom) Supplementry Figure 1 Mouse model of bldder cncer. () Schemtic digrm describing mouse model of BBN-induced bldder cncer. (b) Histopthologicl nlysis (H&E) of invsive crcinom fter 6 months of BBN exposure from three different nimls. Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

2 SUPPEMENTARY INFORMATION Bldder exposed to 5 weeks Ki67/minin/DAPI CK5/minin/DAPI Supplementry Figure ck of urothelil prolifertion t erly stges of BBN exposure. Seril sections mde from bldder exposed to 5 weeks were stined with Ki67 (left pnel, green) nd CK5 (right pnel, green). Sections were co-stined with lminin (red) nd DAPI (blue). Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

3 SUPPEMENTARY INFORMATION Shh CreER/WT ; R6 mtmg/wt ( 4 months; TM t month) b Shh CreER/WT ; R6 mtmg/wt ( 6 months; TM t month) H&E me/m H&E me/m Bldder #1 Bldder #1 Bldder #3 Bldder #3 Bldder #5 Bldder # Bldder # Bldder #4 Bldder #4 Supplementry Figure 3 Mrked Shh-expressing bsl stem cells give rise to CIS nd invsive crcinom. () Shh CreER ; R6 mtmg mice (four different nimls, bldder #1-4) injected with TM on three consecutive dys were exposed to 4 months to induce development of CIS nd bldder tissues nlyzed (H&E, left pnel; mg/mt expression, right pnel). (b) Shh CreER ; R6 mtmg mice (five different nimls, bldder #1-5) injected with TM on three consecutive dys were exposed to 6 months nd bldder tumors nlyzed (H&E, left pnels; mg/mt expression, right pnels)., bldder lumen. Representtive imges re shown in Figure. Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

4 SUPPEMENTARY INFORMATION TM t month; 8 months Shh CreER/WT ; R6 DTA/WT No TM; 6 months Bldder #1 Bldder #7 Bldder # Bldder #8 Bldder #4 Bldder #1 Bldder #5 Bldder #3 Bldder #9 Bldder #11 Bldder #6 Bldder #1 Supplementry Figure 4 Abltion of Shh-expressing bsl cells renders bldder resistnt to nitrosmine-induced formtion of invsive crcinom. TM ws injected into Shh CreER ; R6 DTA mice (twelve different nimls; bldder #1-6, with TM; bldder #7-1, without TM) on five consecutive dys to blte Shh-expressing bsl cells, nd these mice subsequently were exposed to 6 months (right pnels; without TM) nd 8 months (left pnels; with TM). Bldder tissues were nlyzed by H&E stining., bldder lumen. Representtive imges re shown in Figure 3c. Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

5 K K 15K 1K 5K 5K K 15K 1K 5K 5K K 15K 1K 5K K 15K 1K 5K 5K 5K K 15K 1K 5K 5K K 15K 1K 5K K K 15K 1K 5K 5K K 15K 1K 5K 5K K 15K 1K 5K 5K K 15K 1K 5K 5K K 15K 1K 5K 5K K 15K 1K 5K SUPPEMENTARY INFORMATION Bldder #1 H&E Shh CreER/WT ; R6 mtmg/wt ( 4 months; TM t 4 months) me CK5(pseudo) Merge b Tumor # Totl me+ m Tumor # Bldder # Tumor # Bldder #3 Tumor # Bldder #4 Tumor # Tumor # c Tumor injection into the wll of bldder from immunocompromised mouse Orthotopic model of BBN induced tumor d 1 5 cells 1 4 cells 1 3 cells Orthotopic llogrfts of me positive BBN induced tumor (Shh CreER/WT ; R6 mtmg/wt ; 6 months; TM t 4 months) Orthotopic llogrfts of m positive BBN induced tumor (Shh CreER/WT ; R6 mtmg/wt ; 6 months; TM t 4 months) Tumor # Tumor #3 Tumor # Tumor #3 1 5 cells 1 4 cells 1 3 cells Supplementry Figure 5 Tumor-propgting cells derive exclusively from Shh-positive cells in the CIS lesion. () Shh CreER ; R6 mtmg mice (four different nimls: Bldder #1-4) were exposed to 4 months to induce development of CIS. TM ws injected on three consecutive dys to lbel Shhexpressing CIS cells prior to scrifice nd nlysis of bldder tissues by H&E stining (left pnel) or by immunostining for mg nd CK5 (green nd red, respectively, in three right pnels)., bldder lumen. (b) mg/epcam-positive nd mt/epcam-positive popultions from six different invsive crcinoms generted s described in Fig. 5b were seprted using FACS. (c) BBNinduced bldder tumor cells were injected intrmurlly into the dome of the bldder. (d) Orthotopic trnsplnttion with seril dilutions of mg/epcampositive nd mt/epcam-positive cells from invsive crcinoms # nd #3 (invsive crcinom # 1 is shown in Fig. 5e). Representtive imges for () nd (d) re shown in Figure 5, nd 5e, respectively. Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

6 SUPPEMENTARY INFORMATION Supplementry Figure 6 Norml urothelium 4 months CIS (TM injection) months Shh CreER/WT ; R6 mtmg/wt Green: Shh+, CK5 high Red: Shh-, CK5 high Invsive crcinom FACS c Norml urothelium 4 months Shh CreER/WT ; R6 mtmg/wt CIS (TM injection) months Green: Shh+, CK5 high Red: Shh-, CK5 high Invsive crcinom FACS NOD/SCID/IRg null Allogrft b Allogrfts of E or td positive BBN induced tumor (Shh CreER/WT ; R6 mtmg/wt ; 6 months; TM t 4 months) Trnsplnttion of positive cncer cells td positive cncer cells Trnsplnttion of E positive cncer cells E positive cncer cells NOD/SCID/IRg null d Anlysis NOD/SCID/IRg null td positive cncer cells Allogrft E positive cncer cells Allogrft of mixed cncer cells contining two different popultions (Shh CreER/WT ; R6 mtmg/wt ; 6 months; TM t 4 months) H&E E/DAPI td/dapi Mix Supplementry Figure 6 Shh-negtive cells in CIS lesion do not contribute to tumor-propgting cells in invsive crcinom. () Shh CreER ;R6 mtmg mice were exposed to 4 months to induce CIS lesions, then injected with TM on three consecutive dys, which heritbly lbels Shh-positive cells with E wheres cells tht do not express Shh remin lbeled with td. Mice were subsequently treted with two more months, nd E nd td positive cncer cells in invsive crcinom from the resulting nimls were seprted by FACS. E nd td positive cells were then trnsplnted subcutneously into immunocompromised mice (NOD/SCID/ IRg null ). (b) Allogrfts from trnsplnttion of td- nd E-positive cells re shown in left nd right pnels, respectively. (c) Experimentl strtegy to investigte the tumorigenic cpcity of mixed cncer cells originting from Shh-positive or -negtive CIS cells. Shh CreER ; R6 mtmg mice were treted with 4 months to induce CIS, then injected with TM on three consecutive dys to mrk Shh-positive nd -negtive cells with E nd td, respectively. Mice were subsequently treted with two more months, nd E- nd td-positive cncer cells from the resulting nimls were seprted by FACS. Equl numbers of E- nd td-positive cells were then mixed nd subcutneously trnsplnted into immunocompromised mice (NOD/SCID/IRg null ). (d) Allogrft tumor from the experiment described in (c) ws nlyzed by H&E stining (left pnel) nd immunostining for E nd td (green nd red, respectively in middle nd right pnels). Note only E, not td, is expressed in the tumor llogrft. Scle br represents 5μm Mcmilln Publishers imited. All rights reserved.

7 SUPPEMENTARY INFORMATION Invsive crcinom ( 6 months) Before CM Tumor #1 Tumor # Tumor #3 After CM Before CM After CM Before CM After CM b Are # Are #3 Expression of Shh (reltive to HPRT) Shh trnscripts ND ND ND ND ND ND ND ND Tumor #1 Tumor # Tumor #3 Bsl cells Are Are 3 Are 1 Are Are 3 Are 1 Are Are 3 Are #3 Are # Are #1 Are #3 Are # Are #1 Supplementry Figure 7 oss of Shh expression in invsive crcinom. () ser cpture microdissection of three different tumor res from three distinct bldder tumors. Nine tumor res were ssessed; 3 different tumor res from 3 distinct tumors. Representtive imges (re #1 from tumor #1) re shown in Figure 6c. (b) Anlysis of Shh mrna expression by qrt-pcr in microdissected bsl urothelium nd crcinom cells. ND, not detected. Dt re presented s men ± s.e.m from 3 technicl replictes; 9 tumor res were ssessed (3 different tumor res from 3 distinct tumors). Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

8 SUPPEMENTARY INFORMATION Actin CreER/WT ; R6 Rinbow/WT (Bldder t month; TM t month) b Actin CreER/WT ; R6 Rinbow/WT (Intestine t 4 months; TM t month) Supplementry Figure 8 Stochstic four color fluorescence mrking of norml bldder nd intestinl cells. () TM ws injected into Actin CreER ; R6 Rinbow mouse to lbel ll cells in the bldder with one of four fluorescence colors prior to BBN exposure. Right pnels show mgnified views of the regions highlighted by white boxes in the left pnel. (b) Mouse intestine 4 months fter TM injection into Actin CreER ; R6 Rinbow mouse. Note clonl expnsions of intestinl stem cells from crypts into the villi, s expected, thus vlidting fourcolor mrking with the Rinbow mouse., lumen. Scle brs represent 5μm Mcmilln Publishers imited. All rights reserved.

9 SUPPEMENTARY INFORMATION Supplementry Tble 1 Histopthology of BBN- exposed bldder Smple number Time of BBN exposure 1month months 3months 4months #1 No CIS No CIS No CIS Widespred CIS (>3%) # No CIS No CIS No CIS Widespred CIS (>3%) #3 No CIS No CIS Focl CIS (<1%) Scttered CIS (1-3%) #4 No CIS No CIS Focl CIS (<1%) Scttered CIS (1-3%) #5 N/A N/A Scttered CIS (1-3%) Widespred CIS (>3%) #6 N/A N/A Widespred CIS (>3%) Widespred CIS (>3%) #7 N/A N/A Scttered CIS (1-3%) Widespred CIS (>3%) Mcmilln Publishers imited. All rights reserved.

10 SUPPEMENTARY INFORMATION Supplementry Tble Histopthology of BBN- exposed bldder from vehicle- treted Shh CreER ; R6 DTA mice Smple number Time of BBN exposure 1month months 3months 4months #1 No CIS No CIS No CIS Scttered CIS (1-3%) # No CIS Focl CIS No CIS Scttered CIS (1-3%) #3 No CIS No CIS Focl CIS (<1%) Scttered CIS (1-3%) #4 No CIS No CIS Focl CIS (<1%) Widespred CIS (>3%) #5 N/A N/A Focl CIS (<1%) Widespred CIS (>3%) #6 N/A N/A Scttered CIS (1-3%) Widespred CIS (>3%) #7 N/A N/A Scttered CIS (1-3%) Widespred CIS (>3%) #8 N/A N/A Scttered CIS (1-3%) Widespred CIS (>3%) Mcmilln Publishers imited. All rights reserved.

11 SUPPEMENTARY INFORMATION Supplementry Tble 3 Histopthology of BBN- exposed bldder from tmoxifen- treted Shh CreER ; R6 DTA mice Smple number Time of BBN exposure 1month months 3months 4months #1 No CIS No CIS No CIS No CIS # No CIS No CIS No CIS No CIS #3 No CIS No CIS No CIS No CIS #4 No CIS No CIS No CIS No CIS #5 N/A N/A No CIS Widespred CIS (>3%) #6 N/A N/A Focl CIS (<1%) Widespred CIS (>3%) #7 N/A N/A No CIS Widespred CIS (>3%) #8 N/A N/A Focl CIS (<1%) No CIS #9 N/A N/A N/A No CIS Mcmilln Publishers imited. All rights reserved.

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