Winlist 3d version 8

MG63, SAOS2, and ZK58 cells were seeded in 6-well plates and treated for 48 h the next day with 5 (MG63 and SAOS2) or 60 nM (ZK58) of FK866, or 0.1% DMSO. Drug dose was defined as the IC75 determined in a 72 h exposure in 2D. Adherent cells were trypsinized and collected with supernatant. Cells were centrifuged, washed with PBS, and fixed in cold methanol for at least 20 min, as described in [45 (link)]. Cells were stained for 30 min with DAPI in PBS/1% Bovine serum albumin/0.05% Tween and stored at 4 °C overnight. At least 10,000 single cell events were measured with the NucleoCounter NC-250 (Chemometec, Lillerød, Denmark), and analyzed using Winlist 3D Version 8, and Modfit Version 4.1.7 (Verity Software House, Topsham, ME, USA).

CH2879, JJ012, and SW1353 cells were seeded in 6 well plates (9 × 10⁴ to 21 × 10⁴ cells/well). After overnight attachment, cultures were treated with 500 nM talazoparib or the solvent control DMSO for 24 h or 48 h. After the indicated treatment times, adherent cells and supernatant were collected in one sample. Cells were counted, fixed, and washed as described before [58 (link)]. Cells were stained with 4 µM 4’,6-diamidino-2-fenylindool (DAPI) in PBS/1% Bovine serum albumin (BSA)/0.05% Tween20 for 30 min at room temperature and stored at 4 °C overnight. Readout and analysis were performed with the NucleoCounter NC-250 (Chemometec, Denmark), Winlist 3D Version 8, and Modfit Version 4.1.7 (Verity Software House, Topsham, ME, USA). Each analysis measured >10,000 single cell events with a coefficient of variation (CV)lower than 4. A statistical model with a polynomial S-phase shape showed the best fit (Reduced chi-square (RCS) between 1 and 3). Experiments were repeated three times.