Multicycle

Biopsies were minced in the presence of NST buffer and DAPI according to published protocols [23 (link), 39 (link), 40 (link)]. Prior to sorting each sample was filtered through a 35 um mesh and collected into a 5 ml Polypropylene round bottom tube. The mesh was rinsed with an additional 750 ul of NST/10% fetal bovine serum and placed on ice while processing remaining samples. The total volume in the tube for each sample was approximately 1.5 ml. An equal volume of 20 ug/ml DAPI was added to each tube to achieve a final concentration of 10 ug/ml DAPI for flow sorting with a BD Influx cytometer with ultraviolet excitation (Becton-Dickinson, San Jose, CA). The optimal settings for sorting FFPE samples with the Influx sorter were as follows: Drop formation was achieved with piezzo amplitude of 6–10 volts and a drop frequency of 30 khertz. The sort mode was set to purity yield with a drop delay of 31.5–32. Sheath fluid pressure was typically 17–18 psi with a 100 um nozzle. For single parameter DNA content assays DAPI emission was collected at >450 nm. DNA content and cell cycle were then analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).

The tumor was dissociated, and flow cytometry was used to separate and collect nuclei from aneuploid epithelial tumor cells^{38 (link)}. Tissue was minced with scalpels in a solution of 10 µg/ml 4,6-diamidino-2-phenylindole (DAPI) and 0.1% Nonidet P-40 detergent in a Tris-buffered saline. The supernatant was triturated with a 26-gauge needle, filtered through 40 µm steel mesh, and analyzed using an InFlux cytometer (Cytopeia Beckton-Dickenson, Seattle WA), with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content was analyzed MultiCycle (Phoenix Flow Systems, San Diego, CA). 50k aneuploid nuclei were collected in nuclei storage buffer (50 mM Tris·Cl, pH 8.0, 5 mM MgCl₂, 40% Glycerol, 0.1 mM EDTA) and snap-frozen for ATAC-seq.

Cell cycle distribution was analyzed via flow cytometry as previously described (26 (link), 27 (link)). In short, MDA-MB-231 cells treated with PDCe were washed with PBS and then treated with 5% trypsin, then fixed in 75% ethanol overnight at −20°C. Subsequently, the fixed cells were stained with PI in darkness. The stained cells were analyzed using an FC-500 flow cytometer (Beckman Coulter, Miami, FL). Cell cycle distribution was analyzed using the software Multi-Cycle (Phoenix Flow Systems, San Diego, CA). The proportion of cells in G0/G1, S, and G2/M phases was represented in the DNA histogram. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. A total of 10,000 events were recorded in each experimental sample.