br Cell culture br The human colorectal cancer
The human colorectal cancer cell lines, HCT-15, HCT-116, SW620, DLD1, HT-29, and SW480, were purchased from American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 (HCT-15, DLD1), McCoy's 5A (HCT-116), or L-15 (SW620) containing penicillin (100 U/ ml), streptomycin (100 μg/ml), sodium pyruvate (1 mM), and 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit-Haemek, Israel) and maintained at 5% CO2 and 37 °C in a humidified incubator. All Apratastat were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and maintained in culture for a max-imum of 8 wks.
In vitro kinase assays
MBP or ERK1 was used as the substrate for an in vitro kinase assay with 100 ng of active TOPK or MEK1. Reactions were conducted in 1X kinase buﬀer (25 mM Tris–HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, and 5 mM MnCl2) containing 100 μM ATP at 30 °C for 30 min. Reactions were stopped by adding 2X protein loading dye and proteins detected by Western blotting.
In vitro and ex vivo pull-down assays
serum albumin). After incubation with gentle rocking overnight at 4 °C, the beads were washed 3 times with buﬀer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, and 0.01% NP-40), and binding was visualized by Western blotting. For the ATP competitive binding assay, active TOPK was incubated with 3-DSC-Sepharose 4B beads with ve-hicle, 10, 100, or 1000 μM ATP and binding was visualized by Western blotting.
To confirm whether 3-DSC could bind with TOPK, we performed in silico docking using the Schrödinger Suite 2016 software programs (Schrödinger, 2016). The TOPK structure was built with Prime followed by refining and minimizing loops in the binding site. The structure was then prepared under the standard procedures of the Protein Preparation Wizard (Schrödinger Suite 2016). Hydrogen atoms were added with a pH consistent with 7 and all water molecules were removed. The TOPK ATP-binding site-based receptor grid was generated for docking.
Cell proliferation assay
Cells were seeded (1 × 103 cells per well) in 96-well plates and incubated for 24 h and then treated with diﬀerent doses of 3-DSC. After incubation for 24, 48, or 72 h, 20 μl of 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT, Ruitaibio, Beijing, China) were added, and then cells were incubated for 1 h at 37 °C in a 5% CO2 in-cubator. The supernatant fraction was then discarded and 100 µl of dimethyl sulfoxide (DMSO, ≥99.7%, Sigma-Aldrich Co. LLC) were added to dissolve the formazan crystals. Absorbance was measured at 570 nm. For anchorage-independent cell growth assessment, cells (8 × 103 per well) were suspended in media containing 10% FBS for cell maintenance. Then 0.3% agar with vehicle, 5, 10, or 20 μM 3-DSC was added to each cell line in a top layer, over a base layer of 0.5% agar with vehicle, 5, 10, or 20 μM 3-DSC. The cultures were maintained at 37 °C in a 5% CO2 incubator for 2 or 3 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v.6.1) program (Media Cybernetics, Rockwille, MD).
Cell cycle analysis
Cells (2 × 105) were seeded into 60-mm dishes and cultured over-night at 37 °C in a 5% CO2 incubator. To examine cell cycle under normal culture conditions, cells were treated for 48 h with the indicated concentrations of 3-DSC in complete cell culture medium. Cells were trypsinized, fixed overnight and then stained with propidium iodide (20 µg/ml) for 15 min at 4 °C. The cell cycle distribution was measured by FACScan flow cytometry (BD FACS Calibur flow cytometer).
Apoptosis in the presence or absence of 3-DSC was examined using flow cytometry by staining the cells with annexin V-FITC and propi-dium iodide (BioLegend, Nagoya, Japan). Cells (2 × 105) were seeded into 60-mm dishes and cultured at 37 °C in a 5% CO2 incubator. After treatment with 3-DSC for 72 h, cells were harvested and stained with annexin V-FITC and propidium iodide, and then analyzed by FACScan flow cytometry.
Western blot analysis
Cells were disrupted on ice for 30 min in lysis buﬀer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM so-dium vanadate, and 1 mM phenylmethylsulfonyl-fluoride). After cen-trifugation at 14,000 rpm for 15 min, the supernatant fractions were harvested as the total cellular protein extracts. The protein
concentration was determined using a protein assay kit (Solarbio Life Science, Beijing, China). The total cellular protein extracts were sepa-rated by SDS-PAGE and transferred to polyvinylidene fluoride mem-branes in 20 mM Tris–HCl (pH 8.0) containing 150 mM glycine and 20% (v/v) methanol. Membranes were blocked with 5% nonfat-dry milk in 1X PBS containing 0.05% Tween-20 (PBS-T) and incubated with antibodies against pTOPK, TOPK, pMEK1/2, MEK1/2, pERK1/2, ERK1/ 2, pRSK2, RSK2, pc-Jun, total c-Jun, p21, PARP, caspase-3, caspase-7, cleaved PARP, cleaved caspase-3, cleaved caspase-7, or β-actin. Blots were washed 3 times in 1X PBS-T buﬀer, followed by incubation with the appropriate horseradish peroxidase-linked immunoglobulin G (IgG). The specific proteins in the blots were visualized using an en-hanced chemiluminescence detection reagent and the Amersham Imager 600 (GE Healthcare life Science, Pittsburgh, PA, USA).