Countess automated counter

PB samples were obtained for pharmacodynamic studies from patients with
≥20% blood blasts during Cycle 1 on Day 1 before treatment, then Day 5
one hour after completion of decitabine infusion, Day 8 any time, and Day 29
before Cycle 2 treatment. For patients receiving 10-day decitabine, sampling was
on Day 1 before treatment, and Days 5, 8 and 10 one hour after completion of
decitabine infusion, then 29. BM samples were collected at screening and on Day
25–29 of Cycle 1, before Cycle 2 treatment. A schematic of treatment and
sample collection for the talazoparib and 5- and 10-day decitabine regimens is
shown in Supplementary Figure
S1.
Mononuclear cells (MNCs) were isolated from AML samples by density
centrifugation over Ficoll-Paque (Sigma-Aldrich, St Louis, MO). Cells were
counted on the Countess automated counter (Thermo Fisher Scientific, Waltham
MA). MNCs were viably cryopreserved in 95% fetal bovine serum (FBS) and 5%
dimethylsulfoxide for genome-wide DNA methylation analysis, the proximity
ligation assay and measurement of γH2AX foci. MNCs were also centrifuged
at 1500 rpm for 5 minutes and cell pellets were flash-frozen in liquid nitrogen
for the HR activity assay and for RNA sequencing.

Low-passage immortalized human bronchial epithelial cells (BEAS-2B; ATCC) were cultured in DMEM supplemented with 5% FBS, 1% L-glutamine (Corning), and 1% penicillin/streptomycin (Life Technologies). Cell cultures were maintained in 100 mm dishes (Corning) and kept in a humidified atmosphere of 37 °C and 5% CO₂. Regular passaging of cells was performed using 0.25% trypsin/ethylene diamine tetraacetic acid (EDTA) solution (Life Technologies).
Half-maximal inhibitory concentration (IC₅₀) values for each nanoclay and its byproduct were determined using BEAS-2B cells seeded at a density of 1.5 × 10⁵ cells/mL into 12-well plates (Thermo Scientific) and following established protocols.¹ Briefly, confluent cells were exposed to nanoclays or byproducts previously bath sonicated for 10 min (2510 Branson, 100 W) in dispersant DMEM supplemented with 5% FBS. The treatment was performed at doses of 0, 0.03, 0.3, 13, 26, 66, 132, and 197 μg/cm² (0–750 μg/mL) for 24 h. Following exposure, cells were harvested and stained with 0.4% trypan blue solution (Invitrogen). Live cell counts were performed on a Countess automated counter (Invitrogen) to determine IC₅₀ values, which were then used in the remaining assays to compare epithelial monolayer integrity and programmed cell death responses.