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A B Fig. 1C Fig. 2A (G007-LK) Tanaka et al. Supplementary Figure S1 - COLO- 320DM NC CTNNB1 DLD-1 RKO 260K 160K 110K 80K 50K 40K 30K 20K 15K 10K HCT-116 SW403 HCT-15 HCC 2998 KM12 HT-29 GAPDH Active β-catenin Fig. 1C * A COLO- 320DM Phospho β-catenin 40K - + KM12 110K Axin1 160K Axin2 GAPDH G007-LK Active 80K 60K 30K 20K 50K HCT- 116 RKO - + COLO-320DM DLD-1 HCT-15 HCC2998 HT-29 Axin1 76K 38K 102K 150K 225K 24K 31K 52K 17K Active β-catenin 12K GAPDH Axin2 Fig. 2A (G007-LK) G007-LK B 76K 102K 150K 225K - + Phospho β-catenin G007-LK COLO-320DM + - 76K 102K 150K 225K DLD-1 HCT-15 HCC2998 HT-29 Phospho-β-catenin G007-LK SW403 Active β-catenin Phospho G007-LK Axin1 - Axin2 76K 102K 150K 225K 52K 38K 31K 24K 17K + GAPDH Tanaka et al. Supplementary Figure S1

C Axin1 Axin2 102K 76K 38K 31K GAPDH 150K 225K 52K - + HCT15 DLD1 KM12 HCT116 HT29 HCC2998 RKO SW403 IWR-1 COLO-320DM Fig. 2A (IWR-1) Phospho β-catenin 150K 102K 76K Active 52K 38K 31K 24K 17K - + HCT15 DLD1 KM12 HCT116 HT29 HCC2998 RKO SW403 IWR-1 COLO-320DM D Fig. 4A - + siAPC NC HCT-15 DLD-1 HCC2998 G007-LK 102K Axin2 150K 225K 76K APC 52K 38K 31K 24K GAPDH 102K 76K Active β-catenin 150K 225K 38K 31K 24K 17K - + siAPC NC HCT-15 DLD-1 HCC2998 G007-LK Tanaka et al. Supplementary Figure S1 (continued)

A D B C Tanaka et al. Supplementary Figure S2 COLO-320DM Tankyrase Active β-catenin Axin1 Axin2 GAPDH SW403 DLD-1 HCT-15 HCC2998 HT-29 KM12 HCT-116 RKO DLD-1 HCT-15 HCC2998 (High cell density) HT-29 KM12 SW403 Active β-catenin DAPI merge COLO-320DM (Low cell density) B 160K 110K 40K Tankyrase-1 GAPDH siNC siTNKS siTNKS2 COLO-320DM HT-29 Tankyrase-2 C TNKS TNKS2 Relative expression of TNKS Relative expression of TNKS2 NC siTNKS siTNKS2 COLO- 320DM HT-29 COLO- 320DM HT-29 Tanaka et al. Supplementary Figure S2

Cell number (% of control) A Cell number (% of control) TNKSi (μmol/L) G007-LK IWR-1 XAV939 SW480 B 225K 150K 102K 76K 38K 38 K 102 K 225 K 150 K - + NC siAPC APC Axin2 GAPDH Active β-catenin SW480 （20-AAR = 1） COLO-320DM （20-AAR = 0） HT-29 （20-AAR = 3） KM12 （20-AAR = 11） G007-LK Tanaka et al. Supplementary Figure S3

A B C D * ** * ** Tanaka et al. Supplementary Figure S4 - + siAPC#1 NC HCT-15 DLD-1 HCC2998 225K 150K 102K 76K 38K APC Active β-catenin Axin2 GAPDH IWR-1 B Relative expression of Axin2 HCC2998 - + NC siAPC#1 siAPC#3 HCT-15 DLD-1 IWR-1 * ** C - + NC siAPC#1 G007-LK * ** HCC2998 HCT-15 DLD-1 Cell number (% of control) D - + IWR-1 HCC2998 HCT-15 NC siAPC#1 siAPC#2 siAPC#3 DLD-1 Cell number (% of control) Tanaka et al. Supplementary Figure S4

Cell number (% of control) A B Endogenous APC APC811 Axin2 Active β-catenin GAPDH 225K 102K 38K - + siAPC HCC2998 IWR-1 1 2 3 4 5 6 7 8 lane # - + siAPC COLO-320DM APC811 IWR-1 102K 38K Axin2 Active β-catenin GAPDH 1 2 3 4 5 6 7 8 lane # C D 225K 102K 38K GAPDH APC811 Endogenous APC - + siAPC HCC2998 Cell number (% of control) G007-LK (μmol/L) IWR-1 (μmol/L) Mock, NC APC811, NC Mock, siAPC APC811, siAPC Tanaka et al. Supplementary Figure S5

TNKSi-sensitive cells TNKSi-resistant cells Axin2 Axin2-dependent degradation of β-catenin “Long” APC that contains two 20-AARs blocks Axin2-dependent degradation of β-catenin. TNKSi-sensitive cells TNKSi-resistant cells “Short” APC APC Repression of Wnt signaling activity Maintenance of High dependency on Wnt signaling Low dependency “Long” APC A C “Short” APC that lacks all 20-AARs has no effect on Axin2-dependent degradation of β-catenin. b-catenin Tankyrase PAR-dependent degradation of Axins TNKSi treatment Accumulation of Axins B Partially retained 20-AARs All seven 20-AARs lost Tanaka et al. Supplementary Figure S6

A B C * * Tanaka et al. Supplementary Figure S7 PDC APC mutation 20-AAR = 0 G007-LK IWR-1 JC-7 R232X ✓ intermediate JC-20 P1483fs sensitive JC-32 W553X JC-33 Q1303X resistant JC-35 I1164fs JC-36 R805X JC-37 T1556fs JC-39 R216X JC-47 F1491fs JC-49 R302X, E1345X JC-52 L1342X, S1343fs JC-54 R876X JC-55 E1306X JC-56 T1023fs, E1554X JC-58 E1295X JC-61 E941X, T1459fs B PDC G007-LK (μmol/L) 0.037 0.11 0.33 1 3 JC-20 83 67 48 47 45 JC-32 94 84 61 55 JC-35 89 71 54 52 JC-39 77 58 56 JC-49 86 24 19 16 JC-58 90 69 59 68 72 JC-7 87 76 85 JC-36 98 80 74 JC-54 79 JC-56 100 97 65 64 JC-33 106 105 111 109 JC-37 99 102 103 JC-47 107 JC-52 104 101 92 JC-55 81 91 95 JC-61 113 PDC IWR-1 (μmol/L) 0.037 0.11 0.33 1 3 9 JC-20 99 97 91 80 58 54 JC-32 93 96 94 74 70 JC-35 85 66 61 JC-39 88 89 82 64 JC-49 103 67 30 21 JC-58 60 78 JC-7 90 76 JC-36 95 JC-54 84 JC-56 106 92 72 JC-61 108 102 79 JC-33 104 111 105 98 JC-37 JC-47 115 JC-52 86 87 JC-55 101 sensitive sensitive intermediate intermediate resistant resistant C 500 1000 1500 sensitive/ intermediate resistant Length of APC (# of amino acids) 20-AAR = 0 20-AAR ≥ 1 * 20-AAR = 0 20-AAR ≥ 1 500 1000 1500 Length of APC (# of amino acids) sensitive/ intermediate resistant * Tanaka et al. Supplementary Figure S7

Cell number (% of control) A JC-35 JC-7 JC-56 JC-49 ** Area of colonies (% of control) sensitive intermediate resistant G007-LK (μM) JC-55 JC-52 B N B 102K 38K Active β-catenin GAPDH JC-35 JC-49 JC-7 JC-56 JC-52 JC-55 C NC CTNNB1 siRNA: Cell number (% of control) sensitive intermediate resistant JC-35 JC-7 JC-56 JC-55 JC-52 JC-49 ** Tanaka et al. Supplementary Figure S8