S trityl l cysteine stlc

To increase microtubule crosslinking by rigor binding Eg5, we treated with 20 μM FCPT (gift of T. Mitchison, Harvard Medical School, Boston, MA) for 15–30 min. Under these conditions, flux was slowed to 0.1 +/− 0.1 μm/min (s.e.m., n=5) vs. 0.8 +/− 0.1 μm/min in control cells (s.e.m.; n=10), but chromosomes still oscillated, indicating that spindle crosslinking was increased but that spindles remained dynamic (allowing us to normalize for centromere stretch over an oscillation cycle as described).
To inhibit Eg5 motor activity, we treated with 5 μM S-trityl-L-cysteine (STLC, Sigma) in MEM for 30 min, and chose cells that remained bipolar, indicating that they entered mitosis before drug treatment began [32 (link)]. Under these conditions, we observed many monopolar spindles, which indicated that the drug treatment effectively inhibited Eg5.
For speckle experiments, we used SiR-tubulin (alongside speckle strategy described above) to visualize microtubules [47 (link)]. We treated with 10 μM verapamil and 100 nM SiR-tubulin (Spirochrome) for 2–6 h to allow time for incorporation of SiR-tubulin into spindles. Under these conditions, there was no detected defect in spindle appearance or behavior.

To analyze the response to defective kinetochore-microtubule attachments, an established assay for error correction in human cells was modified for extracts. The addition of an Eg5 inhibitor, S-Trityl-L-Cysteine (STLC, Sigma Aldrich), results in monopolar spindles with kinetochores in a tension-less, mono-oriented (syntelic) configuration (Houghtaling et al., 2009 (link)). Subsequent removal of Eg5 inhibition allows spindles to bipolarize and chromosomes to align at the metaphase plate through removal of improper kinetochore-microtubules by Aurora B kinase (Lampson et al., 2004 (link)). A range of concentrations of STLC was tested in extracts, and the ratio of monopolar and bipolar spindles generated were recorded in order to determine the IC₅₀ (0.2 µM). In the modified assay for extracts, STLC was added to a final concentration of 0.5 µM at the beginning of metaphase to induce the majority of spindle structures into a monopolar state. After monopolar spindles were generated in metaphase (around 40 minutes), experiments demonstrating error correction could be performed by diluting the STLC-treated extract with ten-fold untreated CSF extract and letting the monopolar spindles re-bipolarize. At the final concentration of 0.05 µM, no monopolar spindles were observed. Samples for Western blot and immunofluorescence were taken 30 minutes after STLC was diluted.

To synchronise in G1, cells were placed in serum-free media for 24 hours. To arrest cells in S-phase, cells were incubated for 16 hours with 2 mM thymidine, released by washing three times with PBS and placed into fresh medium for 6 hours, followed by a second incubation with 2 mM thymidine for 16 hours. Cells in the G2 phase of the cell cycle were obtained 4 hours after release from double thymidine block. Where nocodazole was used to arrest cells in mitosis, treatment was with 50 μg/ml nocodazole (Sigma) for 16 hours. Where Eg5 inhibitor was used to arrest cells in mitosis, cells were incubated with S-trityl-L-cysteine (STLC, Sigma, 5 μM) or monastrol (Tocris, 100 μM) for 16 hours unless otherwise stated. Release from monastrol was achieved by washing cells 3 times in PBS and replacing media. Cdk1 inhibitor (RO-3306, Alexis) was used at 10 μM. Treatment with the Pak inhibitor FRAX597 (a kind gift from Jonathan Chernoff, Fox Chase Cancer Center, Philadelphia, U.S.) was at 2 μM for 6 hours, and with NSC 23766 for 24 hours at 100 μM. For dox-inducible expression of shRNA or Pak2-CFP or Tiam1-HA constructs, cells were treated with doxycyline (1 μg/μl) for 3-4 days (shRNA), or 24 hours (Pak2-CFP, Tiam1-HA constructs).

HeLa, hTERT-RPE1, U2OS, A549, DLD-1, and NIH/3T3 cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin, and 2 mM L-glutamine at 37°C with 5% CO₂. The HEC-6 cell line was cultured in DMEM supplemented with 15% FBS and 2 mM L-glutamine. Doxycycline-inducible cell lines were cultured in medium containing FBS certified tetracycline-free. spCas9 expression in inducible CRISPR/Cas9 cell lines was induced with 1 μg/ml doxycycline hyclate (Sigma) at 24 hr intervals for 2 days. All other doxycycline-inducible constructs were induced with 10 ng/ml doxycycline hyclate, unless indicated in figure legend. Other drugs used on human cells were Nocodazole (Sigma, 330 nM), S-trityl-L-cysteine (STLC; Sigma, 10 μM unless otherwise indicated), Paclitaxel (Taxol; Invitrogen, 1 μM), GSK923295 (CENP-E inhibitor; Selleck Chemicals, 100 nM), proTAME (APC/Ci; R&D Systems, 12 μM), AZ-3146 (Mps1i; Selleck Chemicals, 4 μM), cycloheximide (CHX; Sigma, 50 μg/ml), MG-132 (MG132; Enzo Life Sciences, 10 μM) unless concentration indicated otherwise in figure legend. Cells were enriched in mitosis with treatment with 330 nM Nocodazole for 16-17 hrs or if indicated, 10 μM STLC for 18 hrs. The antibodies used in this study are described in the relevant methods and in Supplementary Table 1.