Facscalibur analyzer

After 24 h following HNK (25 µM) treatment, the cells were harvested and suspended in PBS containing 0.5% BSA for 10 min at room temperature, followed by the addition of 10 μL phycoerythrin-conjugated DCLK1 antibody (Abcam Inc, Cambridge, MA, USA) at room temperature for 1h dark in a rotator. The samples were analyzed using a FACS Calibur analyzer (Becton Dickinson, Mountain, View, CA, USA), capturing 10,000 events for each sample. The results were analyzed with ModFit LT TM software (Verity Software House, Topsham, ME, USA).

A total of 200,000 EAC cells (SK-GT-4 and FLO-1) per well were plated in 6-well plates. After 24 h, EAC cells were treated with IC₅₀ and semi-IC₅₀ concentrations of compounds 6b, 6d, and 6i. After 72 h, EAC cells were washed, resuspended in PBS, and fixed using an ice-cold fixing solution (70% ethanol in PBS), followed by storage overnight at 4 °C. The next day, EAC cells were centrifuged, washed with PBS, resuspended, permeabilized, and stained with FxCycleTM PI/RNase staining solution (Invitrogen). The cell cycle was studied by flow cytometry using an FACS Calibur analyzer (Becton Dickinson, Mountain View, CA, USA). The experimental datasets were plotted using ModFit LT™ software (Verity Software House, Topsham, ME, USA).

BMMCs and BMA were analyzed for the expression of cell-surface antigens with direct three-color analysis using fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated and peridinin chlorophyll protein complex (PerCP)-conjugated monoclonal antibodies as previously reported [33 (link), 34 (link)]. The following antibodies were used for analysis: immunoglobulin G1 control PE (eBioscience, San Diego, CA, USA), immunoglobulin G1 control PerCP (Becton Dickinson, San Jose, CA, USA), immunoglobulin G1 control FITC (BD Bioscience, Franklin Lakes, NJ, USA), anti-CD34-FITC (BD Bioscience), anti-CD34-PerCP (Becton Dickinson), anti-KDR-PE (R&D Systems, Minneapolis, MN, USA), anti CD45-PerCP (BD Bioscience), and anti-CD133–PE (Miltenyi Biotec, Bergisch Gladbach, Germany). The quantification of total mononuclear cells in BMMCs and BMA was determined by Turk’s solution. For FACS analysis, 5 × 10⁵ events were acquired and scored with a FACSCalibur analyzer (Becton Dickinson). Data were processed using the Macintosh CELLQuest software program (Becton Dickinson).

24 h following Quinomycin treatment, cells were subjected to direct immunofluorescence staining followed by flow cytometric analyses. Briefly, the cells were harvested and suspended in PBS containing 0.5% BSA for 10 minutes at room temperature followed by the addition of 10 μl phycoerythrin conjugated DCLK1 antibody (Abcam Inc, Cambridge, MA). The samples were analyzed using a FACS Calibur analyzer (Becton Dickinson, Mountain, View, CA), capturing 10,000 events for each sample. Results were analyzed with ModFit LT^™ software (Verity Software House, Topsham, ME).

Restriction factor saturation assays were performed as previously described [10] (link). Briefly, TE671 cells expressing endogenous human TRIM5alpha were infected with 2-fold serial dilutions of freshly harvested 293T cell supernatants containing LacZ-encoding VLPs. Cultures were incubated for 4–6 hours before adding a fixed amount of GFP encoding N-MLV. After 72 hours, infected cells were harvested and the percentage of GFP positive cells was determined by flow cytometry using a FACS Calibur analyzer (Becton Dickinson).