0.5% Triton X-100 for 5 min at room temperature. Fixed and permeabilized cells were

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1 1 Supplementary Methods Immunohistochemistry EBC-1 cells were fixed in 4% paraformaldehyde for 15 min at room temperature, followed by 0.5% Triton X-100 for 5 min at room temperature. Fixed and permeabilized cells were incubated with 1% bovine serum albumin in PBS for 60 min at room temperature. Next, the cells were incubated with anti- (1:1,000) as the primary antibody overnight at 4 C and Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000) as the secondary antibody with Hoechst and Alexa Fluor 568-conjugated pharoidine for 1 h at room temperature. The cells were observed under an Olympus IX71 fluorescence microscope equipped with a charge-coupled device camera. Additional antibodies phospho-c-met (Y1349), phospho-gsk-3α/β (S21/9), phospho-β-catenin (S33/37/T41), β-catenin (Cell Signaling Technology), and GSK-3 (Upstate). [ 3 H]vinblastine-tubulin binding assay The assay was performed as previously described with some modification (1). Reaction mixtures containing 0.05 mg of porcine brain tubulin (Cytoskeleton) and unlabeled compounds and radiolabeled agents ([ 3 H]vinblastine or [ 3 H]vincristine) in a volume of 0.2 ml of binding buffer were incubated for 2 h at 37 C. The final concentration of 3 H-labeled or unlabeled compounds were indicated in figure. After incubation, aliquots (100 µl) were dropped onto DEAE-cellulose filters (DE81; Whatman); subsequently, the DEAE-cellulose filters were washed five times with ice-cold binding buffer. The washed filters were dried at 37 C, and a scintillation counter (Beckman) was used to measure the radioactivity after applying MICROSCINT-20 (PerkinElmer). 1

2 2 Sub-G1 analysis Cells were treated with indicated concentration of SN-38 or adriamycin for 72 h. After incubation, the cells were harvested and fixed with 70% ice-cold ethanol for 30 min at 4 C. The cells were washed with PBS and subsequently incubated with 0.25 mg/ml RNaseA (Sigma) for 15 min at 37 C; subsequently, propidium iodide was added (final concentration, 50 µg/ml; Sigma), and the cells were incubated for 30 min at 4 C. A Cytomics 500 flow cytometer (Beckman Coulter) with Cytomics RXP and FlowJo software (Tree Star) was used to perform analysis. Supplementary Movie 1-3: Time-lapse imaging of drug-treated EBC-1 cells Fucci-induced EBC-1 cells were cultured for 22 h with control medium (movie 1), 1 µm tivantinib contained medium (movie 2), or 100 nm crizotinib contained medium (movie 3). Cell cycles were monitored by time-lapse fluorescence microscopy. Supplementary Movie 4-6: Time-lapse imaging of drug-treated SK-MEL-28 cells Fucci-induced SK-MEL-28 cells were cultured for 22 h with control medium (movie 4), 3 µm tivantinib contained medium (movie 5), or 30 nm vincristine contained medium (movie 6). Cell cycles were monitored by time-lapse fluorescence microscopy. Reference 1. Natsume T, Watanabe J, Tamaoki S, Fujio N, Miyasaka K, Kobayashi M. Characterization of the interaction of TZT-1027, a potent antitumor agent, with tubulin. Japanese journal of cancer research : Gann. 2000;91:

3 Supplementary Figure S1 K562 K562/VCR CEM CEM/VBL EBC-1 KB3-1 WT KB3-1 MDR1 KB3-1 BCRP KB3-1 MRP1 DLD-1 A549 H460 SK-MEL-28 p-met (Y1234/1235) c-met long c-met short GAPDH IC 50 () low high Supplementary Figure S1: Expression levels of c-met and p-met All the cells used in this paper were lysed and immunoblotted with the indicated antibodies. Cells were in order of lowest to highest IC50 values of tivantinib.

4 Supplementary Figure S2 A Ratio at 22 h control 800 nm 600 nm Red (G 0 /G 1 ) 39.5% 19.6% 18.0% Green (G 2 /M) 50.2% 76.3% 75.8% B 3 µm Vincristine 30 nm 0 h 16 h C Ratio at 16 h 3µM Vincristine 30 nm Red (G 0 /G 1 ) 72.1% 41.6% 42.0% Green (G 2 /M) 20.7% 44.7% 44.0% Supplementary Figure S2: induced G 2 /M cell-cycle arrest similar to vincristine (A, C) The ratio of red fluorescent (G1 arrest) or green fluorescent (G2/M) cells are shown. 22 h (A) or 16 h (C) after drug treatment, Fucci expressing EBC-1 (A) or SK-MEL-28 (C) cells were harvested and analyzed of mag1 (green, G2/M) and mko2 (red, G1 arrest) intensity on a FC 500 flow cytometer. (B) induces G2/M arrest (green) in fluorescent ubiquitination-based cell cycle indicator (Fucci)-induced SK-MEL-28 cells. Fucci-induced SK-MEL-28 cells were cultured with the indicated concentration of tivantinib and vincristine for 20 h. Cell cycles were monitored by time-lapse fluorescence microscopy. Shown are photographs taken at time zero and 16 h after treatment with each drug. White scale bars indicate 100 µm.

5 Supplementary Figure S3 nucleus F-actin merge control (1 µm, 16 h) Crizotinib (100 nm, 16 h) Supplementary Figure S3 : affects microtubule assembly EBC-1 cells were treated with 1 µm and 100 nm crizotinib for 16 h and subsequently fixed and stained with hematoxylin and phalloidin. Confocal images of EBC-1 cells show immunostaining (green), the nucleus (blue), or F-actin (red). White scale bars indicate.

6 Supplementary Figure S4 A549 1 μm SK-MEL-28 1 μm 0 min 15 min 30 min Supplementary Figure S4 : rapidly inhibits microtubule GFP--expressing A549 and SK-MEL-28 cells were treated with the indicated concentrations of tivantinib or vincristine. White scale bars indicate.

7 Supplementary Figure S5 A EBC-1 (24 h treatment) B H460 (24 h treatment) Fresh medium (24 h) Vincristine 300 nm (µm) Paclitaxel 50 nm Fresh medium (24 h) Vincristine 500 nm 10 (µm) colchicine (µm) Paclitaxel 50 nm insoluble insoluble soluble soluble C DLD-1 (24 h treatment) Fresh medium (24 h) Vincristine 500 nm (µm) Paclitaxel 50 nm insoluble soluble Supplementary Figure S5 : inhibits microtubule polymerization in DLD-1 and EBC-1 cells (A, B, C) EBC-1 (A), H460 (B) and DLD-1(C) cells were treated with the indicated concentrations of tivantinib, vincristine, colchicine or paclitaxel. The amount of insoluble tubulin was examined according to the same protocol used for the results in Figure 1C.

8 Supplementary Figure S6 A Body weight (g) EBC1 (200mg/kg, twice/day) B H460 (200mg/kg, twice/day) Days after drug treatment Days after drug treatment C EBC1 D H460 tumor 1 tumor 2 tumor 3 tumor 4 tumor 5 tumor 6 tumor 7 tumor 8 tumor 9 tumor 10 tumor 1 tumor 2 tumor 3 tumor 4 tumor 5 tumor 6 tumor 7 tumor 8 p-met (Y1349) p-met (Y1234/1235) c-met p-β-catenin (S33/37/T41) β-catenin p-gsk-3α/β (S21/9) GSK-3 Supplementary Figure S6: Effect of in vivo (A, B) Nude mice bearing human cancer EBC-1 (A) or H460 (B) cells were treated orally with tivantinib or vehicle control (See Figure 2A, B). Data are represented by the means ± SDs of body weight (g) at each time point (n = 6). (C, D) Tumors resected from nude mice 19 days after drug treatment were lysed in Laemmli Sample Buffer (C) or tubulin detection buffer (D), and immunoblotted with the indicated antibodies. Each tumor (tumors 1, 2, 3 ) was resected from a different mouse.

9 Supplementary Figure S7 A B SPA counts (cpm) SPA counts (cpm) drug (μm) drug (μm) Supplementary Figure S7 : Effects of various small molecule inhibitors on binding of [ 3 H]vincristine or [ 3 H]vinblastine to tubulin. (A, B) Purified tubulin was incubated with 0.3 µm [ 3 H]vincristine (A) or 1 µm [ 3 H]vinblastine (B) and indicated concentrations of each compound at 37 C for 2 h; subsequently, tubulin was trapped onto DEAE-cellulose filters. The amount of [ 3 H]-labeled compound bound to tubulin was determined on a liquid scintillation counter. Each data point represents the mean ± SD of cpm.

10 Supplementary Figure S8 Cluster 2 Cluster 3 Cluster 4 Score= Score= Score= Cluster 5 Cluster 6 Cluster 7 Score= Score= Score= Cluster 8 Cluster 9 Cluster 10 Score= Score= Score= Supplementary Figure S8 : Docking simulation of tivantinib with tubulin Molecular modeling studies using the GOLD docking program were performed to predict the binding mode of tivantinib to the colchicine binding site in tubulin. (magenta) was overlaid on colchicine with tubulin. The top 50 structures calculated by the GOLD program were clustered into 10 groups. Each representative docking pose from each cluster is illustrated. The top scored docking pose is shown in Fig. 3D. The docking score was shown in each panels.

11 Supplementary Figure S9 A B % of apoptotic cells (Annexin V-positive) % of apoptotic cells (sub-g1) 1 µm Vincristine 30 nm ADM 1 µm SN µm Supplementary Figure S9 : Flow cytometric analyses of apoptotic cells (A) The ratio (%) of apoptotic cells in Fig. 6A are shown. (B) Cells were treated with the indicated concentrations of SN-38 or adriamycin for 72 h. Next, the cells were stained with propidium iodide and analyzed on a FC 500 flow cytometer. The ratio of apoptotic cells (sub- G1 population) are shown.