Zm 447439

For synchronization, cells were seeded in media containing 2mM thymidine for 24 hr, released into fresh media for 12 hr, arrested again in 2mM thymidine for 12 hr, released for 8–12 hr, and fixed for immunofluorescence with 2% PFA in PHEM buffer + 0.5% Triton X-100 or ice-cold methanol. For knock-down experiments in stable T-Rex HeLa cell lines lines (Fig. 3, Fig. 4D and Fig. S4), transfection of Ndc80 siRNA (see Key Resource Table for sequence, 75 nM final concentration) was done at first thymidine release and second thymidine block using RNAiMAX (Life Technologies), according to manufacturer’s description. For experiment depicted in Fig. 3E 3.3 µM Nocodazol (Sigma-Aldrich) and 2 µM ZM 447439 (R&D Systems) were added two hours before fixation. For knockdown and replacement experiments (Fig. 4A–C), HeLa cells were co-transfected at the first thymidine release with siRNA oligos (75 nM for Ndc80, 20 nM for Mad2) and 100 ng rescue plasmid using Lipofectamine 2000 (Life Technologies). Cells were transfected a second time with siRNA oligos at the second thymidine block using RNAiMAX (Life Technologies). For mock and siRNA only controls, an empty pEGFP vector was used as the rescue plasmid. For transient transfection Ndc80 tail experiments, 75 nM GAPD siRNA oligos (Thermo Scientific) were included as mock controls.