(A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939 (5 µm)

Supplementary Figure Legends Figure S1. Tankyrase inhibition suppresses cell proliferation in an axin/β-catenin independent manner. (A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939 (5 µm) and the cell numbers were determined at day 6 (n=3). (B) SW480 and 293T β-catenin knockdown or control cells were treated with DMSO or XAV939 (5 µm) and the cell numbers were determined at day 6 (n=3), the knockdown efficiency was validated by western blot. Figure S2. The tankyrase-binding motif of PTEN is required for TNKS1/PTEN interaction. (A) Schematic representation of wild-type and the tankyrase-binding motif deletion mutation and point-mutation of PTEN. The putative tankyrase-binding motif RYQEDG was indicated. (B) 293T cell were co-transfected with constructs encoding myc-tagged TNKS1/TNKS2 together with vector alone or construct encoding SFB-tagged wild-type or tankyrase-binding motif deleted mutant of PTEN (PTEN TBM). IP/Western was conducted using indicated antibodies. (C) 293T cell were transfected with constructs encoding SFB-tagged wild-type or tankyrase-binding motif point-mutant of PTEN (PTEN-AA). IP/Western was conducted using indicated antibodies. Figure S3. PTEN is not PARylated by other PARPs. (A) PTEN is not ribosylated by PARP1 in vitro. Recombinant PTEN and PARP1 were

subjected to in vitro ribosylation assays in the absence or presence of NAD +. The recombinant proteins were detected by the indicated antibodies and the ribosylated proteins were determined with anti-par antibody. (B) The PARylation of PARP1 is not affected by the tankyrases inhibitor XAV939. Recombinant PARP1 were subjected to in vitro ribosylation reaction with the indicated concentration of PARP1/2 inhibitor olaparib and tankyrases inhibitor XAV939 and followed by Western blotting. (C) The ribosylation of TNKS1 and PTEN were diminished by tankyrase inhibitors but not by PARP1/2 inhibitor. The recombinant MBP-PTEN and TNKS1 were subjected to in vitro ribosylaltion reaction with the indicated tankyrases inhibitor XAV939 (5µM), JW55(5µM), Wiki4(2.5µM) and the PARP1/2 inhibitor olaparib(5µm) as described above. (D) Tankyrases inhibitors but nor PARP1/2 inhibitor treatment leads to increased PTEN level. HCT116 cells were treated with the indicated inhibitors for 6 hrs. Cell lysates were analyzed by immunoblotting. (E) The PTEN/TNKS interaction is not affected by DNA damage. HCT116 cells were treated with 5 Gy of ionizing radiation, after 6 hrs, cell lysates were subjected to IP/Western analysis using indicated antibodies. (F) PTEN PARylation is not influenced by DNA damage. 293T cells were transfected with GFP-tagged PTEN, 24hrs after transfection, cells were treated with 5 Gy of ionizing radiation or XAV939 (5µM), and cell lysates were subjected to IP/Western analysis using indicated antibodies after 6 hrs of irradiation or XAV939 treatment. Figure S4. Identify the PARylation sites of PTEN by TNKS1 and TNKS2. (A) Schematic representation of wild-type and the ribosylation sites mutant of PTEN. (B) The ribosylation sites of PTEN is not required for TNKS1/PTEN interaction. 293T cell

were co-transfected with constructs encoding Myc-tagged TNKS1 together with vector alone or construct encoding SFB-tagged wild-type or ribosylation sites point-mutantion of PTEN (PTEN-3A). IP/Western was conducted using indicated antibodies. (C) The ribosylation sites of PTEN is required for PTEN PARylation in vitro. Recombinant MBP- PTEN, MBP-PTEN-3A and TNKS1 were subjected to in vitro ribosylation assays with biotin-labeled NAD +. The recombinant proteins were detected by the indicated antibodies and the ribosylated proteins were determined with anti-biotin antibody. (D) The indicated ribosylation sites of PTEN is required for PTEN PARylation in vivo. 293T cells were transfected with SFB-tagged PTEN or PTEN-3A, 24hrs after transfection, cell lysates were subjected to IP/Western analysis using indicated antibodies. (E) Tankyrases inhibition stabilizes wild-type PTEN but not the ribosylation sites point-mutantion of PTEN. 293T cells transfected with constructs encoding SFB-tagged PTEN or PTEN-3A were treated with tankyrase inhibitor XAV939 (5 µm, 6 hrs). Cell extracts were examined by Western blotting as indicated. Figure S5. The cellular localization of PTEN is not influenced by PTEN ribosylation. (A) PTEN localization is not regulated by TNKS1/2. Hela cells were co-transfected with GFP-tagged PTEN together with myc-tagged TNKS1 or TNKS2, immunofluorescent staining was performed using the anti-myc antibody, and the nuclears were stained by DAPI. (B) Tankyrases inhibition does not change the locolization of PTEN. Hela cells were transfected with SFB-tagged PTEN, 24hrs after transfection, cells were treated with tankyrase inhibitor XAV939 (5 µm, 6 hrs) and followed by immunofluorescence. (C) The wild-type and tankyrase-binding motif point-mutation of PTEN has the same cellular

localization. Hela cells were transfected with SFB-tagged PTEN or PTEN-AA, immunofluorescent staining was performed using the anti-flag antibody. Figure S6. Tankyrases inhibition affects PTEN degradation and ubiquitination. (A) Tankyrases inhibition leads to increased PTEN level in multiple cell lines. SW480, DLD1, RKO and HCT116 cells were treated without or with XAV939 (5 µm) for 6 hrs. Cell lysates were analyzed by immunoblotting using indicated antibodies. (B) Tankyrases inhibition diminishes PTEN ubiquitination. 293T cells were mock transfected or transfected with constructs encoding GFP-PTEN. 24 hrs after transfection, cells were pretreated without or with XAV939 (5 µm) for 6 hrs, and then treated with MG132 (10 µm) or combination of MG132 (10 µm) and XAV939 (5 µm) for another 6 hrs. IP/Western was conducted as indicated to determine in vivo PTEN ubiquitination. Figure S7. PTEN ribosylation is required for PTEN/RNF146 interaction and RNF146 induced PTEN degradation and ubiquitination. (A) Tankyrases inhibition abolishes the PTEN/RNF146 interaction. 293T cell were transfected with constructs encoding GFP-tagged PTEN together with vector alone, constructs encoding myc-tagged RNF146, WWP2, or NEDD4, 24 hrs after transfection, cells were treated with DMSO or XAV939 for 6hrs, Immunoprecipitation reactions were conducted using anti-myc antibody and followed by Western blotting analysis. (B) Double knockdown of TNKS1/2 suppresses PTEN/RNF146 interaction. Lysates form HCT116 TNKS1, TNKS2, TNKS1/2 stable knockdown and control cells were subjected to IP/Western analysis using indicated antibodies. (C) The tankyrase-binding motif of

PTEN is required for its degradation by RNF146. 293T cells were transfected with constructs encoding SFB-PTEN or SFB-PTEN-AA together with constructs encoding myc-tagged WWP2 or RNF146. Western blotting was conducted as indicated. (D) The tankyrase-binding motif (TBM) of PTEN is required for RNF146-mediated PTEN ubiquitination. 293T cells were transfected with the indicated constructs. Cells were treated with 10 µm MG132 for 6 hrs before they were collected. Immunoprecipitation was carried out using anti-flag antibody and Western blotting was performed using indicated antibodies. Figure S8. Identify the Lysine sites required for RNF146 mediated PTEN ubiquitination. 293T cells were transfected with myc-rnf146, HA-ubiquitin and the indicated PTEN constructs. Cells were treated with 10 µm MG132 for 6 hrs before they were collected. Immunoprecipitation was carried out using anti-ha antibody and Western blotting was performed using indicated antibodies. Figure S9. TNKS1/2 downregulation reduces cell proliferation. Immunofluorescent staining of control and TNKS1/2 knockdown HCT116/RKO cells were performed using Ki67 and BrdU antibodies. The nuclei were stained by DAPI. Figure S10. PTEN/TNKS interaction is observed in normal and cancer cell lines. (A) PTEN/TNKS interaction is observed in 10 different colon cancer cell lines. Lysates from the indicated colon cancer cell lines were subjected to IP/Western analysis using

indicated antibodies. (B) PTEN/TNKS interaction is observed in normal breast and breast cancer cell lines. Lysates from the indicated 2 normal breast and 3 breast cancer cell lines were subjected to IP/Western analysis using indicated antibodies. Figure S11. Validation of PTEN, p-akt, Axin1, TNKS1 and TNKS2 specific antibodies for IHC. The TNKS1, TNKS2 and Axin1 antibodies were validated using mock treated HCT116 cells (with low TNKS1, TNKS2, and Axin1 level) and HCT116 cells treated with XAV939 (with high TNKS1, TNKS2, Axin1 level). The PTEN antibody was validated using mocked treated (with low level of PTEN), XAV939 treated (with high level of PTEN) HCT116 cells together with HCT116 cells with PTEN downregulation (shpten) with or without XAV939 treatment (PTEN negative). The p- Akt antibody was validated using mock treated HCT116 cells (with high p-akt level) and HCT116 cells treated with LY294002 (with low p-akt level). Scale bars, 50 µm. Figure S12. Expression of p-akt and PTEN mrna level in human colon tumors. (A) Immunohistochemical (IHC) staining of p-akt and RNAscope for PTEN mrna was shown for the representative normal colon and colon carcinoma specimens on the US Biomax tissue microarrays. Brown staining indicates positive immunoreactivity. Scale bars, 50 µm. (B) p-akt and PTEN mrna expression status in normal colon tissue and colon carcinoma specimens. (C) IHC staining of TNKS1, p-akt and RNAscope of PTEN mrna for representative human colon tumor samples. Scale bars, 50 µm. (D) Correlation between expression status of p-akt/tnks1, p-akt/tnks2, p-akt/pten, TNKS1/TNKS2 in human colon tumor samples.

Table S1. List of PTEN ribosylation sites by TNKS1 and TNKS2. Ribosylation sites of PTEN determined by mass spectrometry analysis were shown for three samples. The score 13 is considered as reliable in pinpointing the site of modification.