*3.5. Cell Cycle Analysis*

CRC cells (2–3 × 105 cells/well) in 6-well plates were treated with PSY for 48 h. Then, the cells were harvested by trypsinization, rinsed twice with cold PBS, and pelleted by centrifugation at 1200 rpm for 3 min. Pellets were re-suspended in 70% ice-cold EtOH for 30 min at 4 ◦C to perform cell fixation. Then, samples were rinsed twice with PBS and stained using a solution containing 100 μg/mL of RNase A, 50 μg/mL propidium iodide (PI) in PBS for 30 min in the dark ([31]). Then, the cell cycle of the treated cells was analyzed using a BD FACS Canto™ II (Franklin Lakes, NJ, USA).

#### *3.6. Apoptosis Analysis*

Apoptosis of CRC cells was analyzed using a fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit (Biovision, Inc. K101-100, Waltham, MA, USA). Cells (2–3 × 105 cells/well) in 6-well plates were treated with PSY for 48 h. After the treatment, both adherent and floating cells were harvested by centrifugation, washed with PBS, and resuspended in 1× binding buffer at 1 × 106 cells/mL. Then, 100 <sup>μ</sup>L of the cell suspension was transferred to a 5 mL culture tube and incubated with 5 μL FITC Annexin V and 5 μL PI for 15 min at room temperature in the dark. Then, 400 μL of 1× binding buffer was

added in the 5 mL culture tube. Fluorescence intensity was analyzed using a BD FACS canto II (Franklin Lakes, NJ, USA). In addition, TUNEL assays were performed using a DeadEnd™ Fluorometric TUNEL assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.

#### *3.7. Immunofluorescence Staining*

Cells were seeded in 8-well chamber slides and treated with PSY for 24 h. After the cells were fixed and permeabilized, the cells were stained with rabbit polyclonal anti-phospho (p)-STAT3 (1:100), Alexa Flour 488 goat anti-rabbit antibody (1:1000) (Invitrogen, Carlsbad, CA, USA) and 4- ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) as described ([31]. Images were obtained using an iRiSTM Digital Cell Imaging System (Logos Biosystems, Anyang, Republic of Korea).

#### *3.8. Transient Transfection and Luciferase (Luc) Assays*

Transient transfection and Luc assays were performed to measure STAT3 activity in PSY-treated cells as previously described ([31]). Cells were transfected with pSTAT3-Luc reporter containing STAT3 binding sites to measure STAT3 activity (Clontech, Palo Alto, CA, USA) in a 60 mm plate using Lipofectamine 2000 (Invitrogen). One day after transfection, cells were re-plated into a 48-well plate. Then, the cells were treated with PSY for 24 h. Luc assays were performed using a Luc assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Luc activity was normalized by protein concentration.
