*2.6. Cell Viability Assay*

The viability of the cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St. Louis, MO, USA). The 2 × 104–4 × 104 cells/mL were seeded in 96-well plates. After the attachment, cells were treated with different concentrations of **B**, EL000327, D, and 5-FU for 48 h in the presence or absence of various inhibitors (Z-VAD-FMK (10 μM) (R&D System, Inc, McKinley Place N.E, MN, USA), 3-MA (1mM), CQ (10 μM), NAC (5 mM) (Sigma-Aldrich, St. Louis, MO, USA), and SP600125 (10 μM) (Cell Signaling Technology, MA, USA). Cells treated with 0.01% of DMSO were used as the control. Next, 15 μL of the MTT reagent was added to each well and incubated for 4 h at 37 ◦C. The medium was aspirated completely and 150 μL of DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells before the absorbance was measured at 540 nm by a microplate reader (Bio Tek Instruments, Winooskim, VT, USA) using Gen 5 (2.03.1) software. SPSS statistical software 23 was used for the IC50 calculation. Synergic effects of **B** with D or 5-FU were assessed by compuSyn software.

#### *2.7. Cell Cycle Analysis by Flow Cytometry*

Caco2, HCT116, DLD1, and HT29 cells were seeded in 6-well plates at the density of 1.5–2 × 105 cells/well, incubated overnight, and treated with 0.01% of DMSO, various concentrations of **B**, and 60 μg/mL of EL000327 for 24 h, 48 h, or 72 h. Cells were harvested and washed with FACS washing buffer, incubated with trypsin solution, followed by RNase A for 10 min at room temperature. Cells were centrifuged and pellets were collected and stained with 100 mL of 4 mg/mL PI (Sigma-Aldrich, St. Louis, MO, USA) for 2 h in the dark at 4 ◦C. Cell cycle analysis was performed on a CytoFLEX instrument (Beckman Coulter Life Sciences, Indianapolis, IN, USA).

#### *2.8. Western Blotting*

Caco2 cells were cultured in 6-well plates at the density of 2 × 105 cells/well overnight and treated with 0.01% of DMSO, different concentrations of **B**, and 60 μg/mL of EL000327 for 24 h or 48 h in the presence or absence of various inhibitors (Z-VAD-FMK (10 μM), 3-MA (1 mM), CQ (10 μM), NAC (5 mM), and SP600125 (10 μM). Cells were harvested and lysed, and the protein concentrations were determined by the BCA protein assay following the manufacturer's instructions. Then, 25 or 50 μg of the total extract was separated by SDS-PAGE (12%) and transferred to a blotting membrane at 1.2 A for 6 h. Membranes were blocked with 5% of skim milk for 1 h followed by incubation with various primary antibodies (Cyclin B1, D1, p-Cdc2, BAX, Bcl-XL, PARP, caspase-3, Beclin 1, P-62, LC3B I/II, p-JNK, JNK, p-Akt, Akt, NF-κB, Actin purchased from Cell Signaling Technology, MA, USA) for 2 h at room temperature (RT). Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 30 to 60 min at RT. Specific antibody binding was detected under chemiluminescence imaging (iBright FL1000 Imaging System, Thermo Fisher Sciences, biomolecular imager, Amersham ImageQuantTM 800 Western Blot Imaging System) and measured by Multi Gauge 3.0. software. Relative density was calculated against the density of the actin bands.

#### *2.9. Tubulin Polymerization Assay*

The effect of **B** on tubulin organization was detected using the Tubulin Polymerization Assay Kit (Cytoskeleton, Inc., Denver, CO, USA) according to the manufacturer's instructions. In brief, tubulin proteins (>99% pure) were suspended at a final concentration of 3.0 mg/mL in ice-cold TP, and the tubulin solution was incubated at 37 ◦C with a general tubulin buffer with or without **B** (3.3, 20, 60 μg/mL). Paclitaxel and vinblastine (10 μM) were used as positive controls for the stabilization or destabilization of the microtubules, respectively. Tubulin polymerization was measured by continuously monitoring the change in turbidity at 340 nm by a microplate reader using Gen 5 (2.03.1) software.

#### *2.10. Quantitative Real-Time PCR*

Total RNA of 0.01% of DMSO, **B** (20, 60 μg/mL), and EL000327 (60 μg/mL) treated Caco2 cells were extracted using RNAiso Plus (TaKaRa, Kusatsu, Shiga, Japan) according to the manufacturer's instructions. cDNA was reverse transcribed from 3 μg of total RNA of each treated group using the M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). mRNA levels of stathmin and MAP4 was measured using stathmin (forward) 5-GGTGGCGGCAGGACTTTCCTTATCCCAGTTGATT-3 and (reverse) 5- TTCTCGTGCTCTCGTTTCTCAGCCAGCTGCTTC-3; MAP4 (forward) 5-CCCTTTCTGAG GTAGCGTGCCTTGTGGAGGT -3 and (reverse) 5-CTGGCTCCCTCATGTTCTTGGCACAG CAGA-3 primers and SYBR green (Enzynomics). qRT-PCR reaction and analysis were performed using CFX (Bio-Rad, Hercules, CA, USA).

#### *2.11. Immunofluorescence (IF) Imaging*

Caco2 cells were cultured on cover slips at the density of 1 × <sup>10</sup><sup>5</sup> in a 12-well plate. After the adherence, cells were treated with 0.01% of DMSO, different concentrations of **B**, paclitaxel (100 nM), vinblastine (50 nM), and deoxyphodophyllotoxin (DPT) (25 nM) for 24 h. Cells were washed with phosphate-buffered saline (PBS) three times, followed by fixation with 4% paraformaldehyde in PBS for 10 min, permeabilization with 0.1% Triton™ X-100 for 10 min at RT, and blocking with 1% BSA in PBS for 1 h at RT. The cells were labeled with alpha tubulin (B-5-1-2) Alexa Fluor 488 Mouse Monoclonal Antibody, at 2 μg/mL in 0.1% BSA, and incubated for 3 h at RT. Cells were washed three times with PBS for 5 min after every step. Then, the cells were blocked again with blocking solution containing 1% BSA for 30–45 min at RT. Cells were stained again with fluorescent phalloidin staining solution and incubated for 30–60 min at RT. After washing with PBS, cover slips were mounted on glass slides with prolong gold with DAPI and left overnight at RT. Images were taken using a K1-Fluo Confocal Laser Scanning Microscope (Nanoscope Systems, Daejeon, Republic of Korea).

#### *2.12. Hoechst Staining*

Caco2, HCT116, DLD1, and HT29 cells were seeded in a 12-well plate containing cover slips at a density of 1 × 105 cells/well. After overnight incubation, cells were treated with 0.01% of DMSO, **B** (20, 60 μg/mL), and EL000327 (60 μg/mL) for 12 or 24 h. Cells were washed with PBS, followed by fixation with 4% paraformaldehyde for 15 min. After washing again with PBS, cells were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 30 min, and stained with Hoechst 33258 (Sigma-Aldrich) for 1 h in the dark at room temperature. Cells were assessed by Nikon Eclipse 400 fluorescence microscope (Nikon Instech Co. Ltd., Kawasaki, Japan) to identify the morphological changers in the nuclei.

#### *2.13. IncuCyte™ Caspase-3/7 and Annexin v Apoptosis Assay*

Caco2 cells were seeded in a 96-well plate at a density of 2.5 × 103 cells/well. Cells were grown overnight to 25–30% confluence at the start of the assay. Media were supplemented with 5 μM of Caspase-3/7 (green) reagent (4440, Essen Bioscience, Morgan Rd, Ann Arbor, MI, USA) or Annexin V (red) reagent (4641, Essen Bioscience, Morgan Rd, Ann Arbor, MI, USA) diluted to 1:200 and added to the cells treated with 0.01% of

DMSO, different concentrations of **B**, and EL000327 for 48 h in the presence or absence of Z-VAD-FMK (10 μM). The apoptosis of cells was determined by fluorescence scanning performed every 2 h for 48 h by the IncuCyte Zoom® instrument with a 10× objective and analyzed with the Standard Scan Type.

#### *2.14. Apoptosis Analysis by Flow Cytometry*

Caco2, HCT116, DLD1, and HT29 cells were cultured in a 6-well plate at the density of 2 × <sup>10</sup><sup>5</sup> cells/well until adherence. Cells were treated with 0.01% of DMSO, different concentrations of **B**, and EL000327 for 48 h in the presence or absence of Z-VAD-FMK (10 μM). Cells were harvested and washed with PBS, resuspended in 100 μL of 1x binding buffer followed by staining with 5 μL of 50 μg/mL propidium iodide (PI; BD Biosciences, San Jose, CA, USA) and 3 μL of Annexin V–FITC (BD, Biosciences, San Jose, CA, USA), for 30 min in the dark. Death cells were detected by flow cytometry on a CytoFLEX instrument (Beckman Coulter Life Sciences, Indianapolis, IN, USA).

#### *2.15. Measurement of ROS Generation*

Caco2 cells were seeded in a 6-well plate at the density of 2 × <sup>10</sup><sup>5</sup> cells/well overnight and treated with 0.01% of DMSO, different concentrations of **B**, and EL000327 for 12 h in the presence or absence of NAC (5 mM). Cells were incubated with DCFH-DA (10 μM) in DMEM medium without FBS for 30 min at 37 ◦C and washed three times with DMEM. ROS generation was determined by fluorescence microscopy (K1-Fluo Confocal Laser Scanning Microscope, Nanoscope Systems, Daejeon, Republic of Korea) and flow cytometry (CytoFLEX; Beckman Coulter Life Sciences, Indianapolis, IN, USA) using peroxide-sensitive fluorescence probe DCFH-DA.
