*2.4. Cell Counting*

MDA-MB-231 cells (10<sup>5</sup> cells) were seeded in 6 cm culture dishes. After attaching overnight, cells were treated with ISL for 48 h. Cell morphologies were photographed using a light microscope (Olympus, Tokyo, Japan). Then, cells were detected by trypsinization and resuspended in the culture medium. Cell suspensions were mixed with 0.4% trypan blue solution (Gibco, Grand Island, NY, USA). The number of cells was counted using a hemocytometer under an inverted phase-contrast microscope at 200× magnification.

#### *2.5. Flow Cytometry Analysis of Cell Cycle Distribution and Apoptosis*

To analyze the cell cycle distribution, MDA-MB-231 (1 × 105) cells were seeded into 6 cm culture dishes. After attaching overnight, cells were treated with the vehicle or ISL at 25 and 50 μM in DMEM/F12 medium containing 10% FBS for 48 h. At the end of incubation, cells were detached by trypsinization. For the cell cycle distribution assay, the cells were fixed in 70% alcohol at −20 ◦C and stained with a propidium iodide (PI) solution (2 mg of DNAse-free RNAse A and 0.4 mL of 500 μg/mL PI was added to 10 mL of 0.1% Triton X-100 in phosphate buffered saline (PBS)) at room temperature for 30 min. For the apoptosis analysis, a commercial annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was used. The procedure was performed according to the manufacturer's protocols. The cell cycle distribution and apoptosis were analyzed using a BD FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). A minimum of 10,000 cells per sample were collected and further analyzed using CellQuest software (BD Biosciences).

#### *2.6. Protein Preparation and Western Blot Analysis*

Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Baden-Württemberg, Germany). Protein was quantitated by the Bradford assay and then resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a polyvinylidene Fluoride (PVDF) membrane, 1.5% Bovine serum albumin (BSA) solution was used

to block the empty space of the membrane. Subsequently, membranes were incubated with primary antibodies—CDK4 (1:1000; Cell Signaling, Boston, MA, USA), cyclin D1 (1:1000; Cell Signaling), Bcl-2 (1:1000; Cell Signaling), Bax (1:1000; Cell Signaling), caspase-3 (1:1000; Cell Signaling), PARP (1:1000; Cell Signaling), GAPDH (1:10,000; Proteintech, Rosemont, IL, USA), p62 (1:1000; Cell Signaling), LC3 (1:1000; Cell Signaling), Beclin1 (1:10,000; Novus Biologicals, Littleton, CO, USA), Caspase-8 (1:1000; Cell Signaling), and Cathepsin B (1:1000; Cell Signaling) and β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA)—at 4 ◦C overnight and then with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 to 2 h. The visual signal was captured by an image analysis system (UVP BioChemi, Analytik Jena US, Upland, CA, USA). The band densities were determined as arbitrary absorption units using the Image-J software program Version 1.52t (NIH, Bethesda, MD, USA). The expression level of these target proteins was analyzed by three individual experiments. In the same quantitative protein sample, two di fferent molecular weights of the target protein were determined by the same gel. The membrane was cut into two fractions and incubated with two di fferent antibodies at the same time.
