*3.2. Biological Assays*

#### 3.2.1. In Vitro MTT [3-(4,5-Dimethylthiazaol-2-yl)-2,4-diphenyltetrazolium bromide] Assay

Cell viability was evaluated by measuring the reduction in MTT to yield blue formazan. Cells were cultured in 96-well plates, allowed to attach overnight, and then treated with compounds. After treatment, MTT solution (1 mg/mL) was added to each well, and plates were incubated for another 2 h. Medium was removed, blue formazan was dissolved in DMSO, and the absorbance was read at 570 nm by Multiskan Go spectrophotometer (Thermo Scientific, Madison, WI, USA).

#### 3.2.2. In Vivo Antitumor Activity Assay

Male nu/nu mice (5 weeks old) from National Laboratory Animal Center (Taipei, Taiwan) were maintained under the procedures and guidelines provided by the Institutional Animal Care and Use Committee of the National Health Research Institutes (Taipei, Taiwan). All experiments were supervised under the Institutional Animal Care and Use Committee, China Medical University, Taichung, Taiwan with a protocol number (CMUIACUC-2018-278). HCT-116 colon cancer cells (5 <sup>×</sup> 106 cells per mouse) were suspended in 0.1 mL of Matrigel solution (50% *v*/*v* Matrigel in PBS) and inoculated into the mammary fat pads of the mice. When the tumor size reached 100 mm3, the tumor-bearing mice were randomly divided into four groups for treating with vehicle (5% ethanol and 10% tween 80 in 85% saline, 0.1 mL) and **2** (p.o. 50, 100 and 150 mg/kg/day body weight) respectively. Compounds was administered via oral route daily for 30 days. 5-Fluorouracil-treated group (i.p. 30 mg/kg) is the

positive control. Tumor size and mouse body weight were measured once every 3 days, and tumor volume (mm3) was calculated using the equation: length <sup>×</sup> (width)2 <sup>×</sup> 0.5. At the end of experiments, mice were sacrificed.
