*2.7. Cell Transfection*

The siRNA of SQSTM1/p62 (SignalSilence ® SQSTM1/p62 (Cell Signaling) were transfected into MDA-MB-231 cells with lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) reagen<sup>t</sup> according to the manufacturer's instructions. Briefly, 100 nM sip62 was transfected into MDA-MB-231 and treated with 50 μM ISL for 48 h.

#### *2.8. Tumor Xenograft Mouse Model*

The animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (permit number: 99-190-C). In this study, 5-week-old female Nude-Foxn1nu mice were purchased from BioLASCO (Taipei, Taiwan). The mice were housed with 12 h of artificial illumination in a temperature-controlled room (22 ± 2 ◦C). Food and water were provided ad libitum. Mice were randomly divided into two groups: (1) mice treated with PBS (0.25 mL/mouse) by oral gavage once daily for 2 weeks as a control, and (2) mice pretreated with ISL (Sigma-Aldrich) at 2.5 or 5.0 mg/mL by oral gavage (0.25 mL/mouse) once daily for 2 weeks. After pretreatment for 2 weeks, mice were inoculated with 100 μL PBS/mouse only as the control or human breast MDA-MB-231 cancer cells (5 × 10<sup>6</sup> cells in 100 μL PBS/mouse) by a subcutaneous (s.c.) injection on the right flank, and they were constitutively treated with ISL for an additional 25 days. After implantation for 1 week, tumor volume was measured using calipers and the following formula was calculated: 0.5 × length × width2. At the end of the experiment, the mice were sacrificed by an intraperitoneal (i.p.) injection with an anesthetic mixture (30 μL/mouse; Zoletil:Rompun (*v*/*v*) = 1:1). The tumor tissues were weighed, photographed, and then fixed with 10% formalin for further immunohistochemistry (IHC) analysis.
