*2.2. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay*

For assaying cell viability, cells were plated with 3000–4000 cells/well onto a 96-well plate in triplicate and then treated with the indicated amount of TAO for 72 h at 37 ◦C in a 5% CO<sup>2</sup> incubator. After the drug treatment, cells were incubated with 5 mg/mL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, M2128, Sigma-Aldrich, St. Louis, MO, USA) solution in 1X phosphate-buffered saline (PBS) for 4 h in a 37 ◦C incubator. The MTT crystal was dissolved in dimethyl sulfoxide (DMSO, D1370, Duchefa Biochemie, Haarlem, The Netherlands), and cell viability was measured at 540 nm by spectrometry.

#### *2.3. Western Blot Analysis*

Cells were plated with 200,000 cells/well on a 6-well plate. The cells were treated with TAO at concentrations indicated in the text for 48 h. Cellular protein was lysed by incubating for 20 min on ice in radioimmunoprecipitation assay (RIPA) buffer containing 1X protease inhibitor mix (P-8340, Sigma-Aldrich, St. Louis, MO, USA) and 1 mmol/L of phenylmethylsulfonyl fluoride (PMSF, P-7626, Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then were electrotransferred to a polyvinylidene difluoride (PVDF) membrane. After blocking membranes with 5% non-fat dry milk in PBS, membranes were incubated with primary antibodies overnight at 4 ◦C. After several washes, blots were incubated with secondary antibodies (GeneTex, Irvine, CA, USA) for 1 h. After an additional wash, light development was initiated by adding enhanced chemiluminescence (ECL) reagents (Amersham PLC, Buckinghamshire, United Kingdom). Primary antibodies for studying phosphorylated Akt (Ser473, #9271), Akt (#9272), and VEGFR2 (#2479) were obtained from Cell Signal Technology (Danvers, MA, USA), and β-actin (sc-47778) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Phosphorylated-vascular endothelial growth factor receptor 2 (VEGFR2, ab5473) and p62 (ab56416) were obtained from Abcam (Cambridge, UK), and anti-LC3 antibody (NB100-2220) was purchased from Novus Biologicals (Centennial, CO, USA).

#### *2.4. ELISA (Enzyme-Linked Immunosorbent Assay) for MMP-2 (Matrix Metallopeptidase 2) and 9*

For the enzyme-linked immunosorbent assay (ELISA), media was collected from the cells in culture and transferred to a 96-well plate for matrix metallopeptidase 2 (MMP-2, MMP200) or MMP-9 (DMP900) specific ELISAs using a Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. A standard curve using each recombinant protein provided in the kit was run with each assay, and the concentration of each protein was determined by the standard curve.

#### *2.5. Caspase-3 Assay*

Cell death was assessed using an active caspase-3 assay kit (KHO1091, Invitrogen, Carlsbad, CA, USA). Cells (2 <sup>×</sup> <sup>10</sup><sup>5</sup> /well) were plated in a 6-well plate and treated with TAO as indicated for 48 h. After TAO treatment, cells were lysed in RIPA buffer, and 50 ug of total protein was used for the caspase-3 assay according to the manufacturer's instructions.
