Cycletest plus kit

GBM cells treated with drugs or DMSO were trypsinized, suspended in ice-cold PBS, and fixed in 70% ethanol at −20 °C. Cell cycle progression was evaluated using BD Cycletest Plus kit and BD FACS Calibur flow cytometer (BD, Franklin Lakes, NJ). After fixing, the cells were washed twice with PBS, stained in 250 μL of trypsin buffer for 15 min, and eventually added to 200 μL of trypsin inhibitor with RNase buffer. The samples were finally stained with 200 μL of PI solution and analyzed.
Cell apoptosis was analyzed using BD annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit. Harvested cells were washed with cold PBS, resuspended in 50 μL of annexin binding buffer, stained with 5 μL of annexin V-FITC and 5 μL of PI solution for 15 min at room temperature in the dark, and then diluted in 400 μL of 1× binding buffer.

Cell cycle was determined with flow cytometry by using BD Cycletest Plus kit (BD Biosciences, USA) following the protocol provided by the manufacturer.

Analysis of cell cycle distribution was performed by flow cytometry. These experiments were done independently at least 3 times and produced similar results. Cells were grown in RPMI1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO₂. A MDA-MB-468 cell culture containing 1 to 2 x 10⁶ cells was treated with the inhibitor (25 or 50 nM PF-7006 or PF-3837) for 96 h. The cells were fixed in 70% ethanol and stained with propidium iodide using the CycleTEST Plus kit (Becton Dickinson). The samples were analyzed on a Becton Dickinson FacsCaliber Instrument. The proportion of cells in G1, S, G2-M, aneuploidy or non-gated (sub-G1) was determined using Cell Quest v.2.0 software (Becton Dickinson). Gates were set based on control cells and used for all of the subsequent sample treatments. For Mps1 treated cells, there is cell death/debris to account for, outside of the subG1 fraction which is picked up with propidium iodide staining. When the cell populations were gated for the experiment, the cellular debris that did not stain well was excluded.

DU145 cells (8 × 10⁵) in supplemented medium were plated into flasks (25 cm²) for 24 h. After incubation, the cells were treated for 24 h by addition of plagiochiline A or vehicle (containing equivalent DMSO) directly to the culture medium to give the final concentration indicated in the figures or legends. After treatment, the cells were harvested by trypsinization. For cell cycle distribution, the cells were fixed, and stained with propidium iodide using the Cycle Test Plus kit (Becton Dickinson, Franklin Lakes, NJ, USA). Three independent experiments were performed, and p-values to assess differences between treatments were calculated using Student’s t-test. For cell death detection, the cells were stained with Annexin-V-FITC and propidium iodide using the Apoptosis Detection Kit (BD Biosciences, Mountain View, CA, USA), according to the manufacturer’s instructions. The cells were then analyzed by flow cytometry using a FACScalibur cytometer (BD Biosciences, Mountain View, CA, USA) and FlowJo program (Tree Star, Inc., Ashland, OR, USA).

After treatment with fucoidan, the cells were harvested with trypsin and washed twice with cold PBS; following this, a DNA reagent kit (Cycle TEST™ PLUS kit, Becton-Dickinson, San Jose, CA, USA) was used according to the manufacturer’s instructions to stain the nucleus. Thereafter, flow cytometry analysis was performed using a flow cytometer (Becton-Dickinson), and the relative DNA content was determined using Cell Quest software (Becton-Dickinson) based on the amount of red fluorescence.