S trityl l cysteine

Mitotic HeLa cells were synchronized in M phase by treating cells with 200 ng/ml nocodazole (Sigma-Aldrich) for 16 h. Cells were then washed and incubated in a proteasome inhibitor, 15 μM MG132 (Sigma-Aldrich), for 3 h followed by collection. For monopolar synchronization, the cells were incubated with an Eg5 inhibitor (5 μM (+)-S-Trityl-L-cysteine (Sigma-Aldrich)) for 16 h and mitotic exit was forced by the addition of 20 μM RO-3306 (Sigma-Aldrich) for 5 min.

PtK1, H2B-PAGFP PtK1, and HEC1-GFP PtK1 [32 (link)] cells were cultured in Ham’s F-12 media (Invitrogen) supplemented with 10% Fetal Bovine Serum (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 14 mM sodium bicarbonate (Fisher Scientific), 1% antibiotic-antimycotic (Invitrogen), and maintained at 37° C in a humidified CO₂ incubator. For live cell imaging, cells were either grown on glass-bottom dishes and imaged using a stage top incubator (Tokai Hit) or were grown on sterilized coverslips inside 35 mm Petri dishes, transferred into a modified Rose chamber [33 (link)] with top coverslip, and imaged on a microscope stage heated by an air stream incubator (Nevtek). To induce LCs and MNi, cells were incubated in 20 μM STLC (S-Trityl-L-cysteine; Sigma-Aldrich) for 3 hours. The drug was then washed out by rinsing the cells 4 times with warm media. Cells were then re-incubated in fresh Ham’s F-12 media for 24 hours before immunostaining or live-cell imaging. For live-cell imaging, cells were placed in phenol-free L-15 media (Gibco) with 4.5 g/liter glucose. To induce DNA damage, 21 hours after STLC washout, cells were treated with 50 μg/ml Bleocin™ (antibiotic from Streptomyces verticillus; Calbiochem) for 3 hours and then fixed and immunostained as described in the next section.

Cells were washed with PBS, lysed in RIPA buffer (50 mm Tris/HCl pH 7.4; 1% NP‐40; 0.5% Na‐deoxycholate; 0.1% SDS; 150 mm NaCl, 2 nm EDTA, 50 mm NaF) containing complete protease inhibitor cocktail (Roche, F.Hoffmann‐La Roche Ltd, Basel, Switzerland) for 30 min on ice, and cleared by centrifugation. Protein concentration was determined using Pierce BCA assay kit (Thermo Fisher Scientific Inc.) with a BSA standard curve. Before loading, protein lysates were denatured at 95 °C for 5 min in 6× SDS sample buffer. Proteins were separated by SDS/PAGE on 7.5 or 10% gels before semi‐dry transfer to 0.45 μm nitrocellulose membranes (GE Healthcare, Chalfont St Giles, UK) and blocked in 5% dry milk powder in TBS‐T (100 mm Tris, pH 7.5, 0.9% NaCl, 0.05% Tween‐20). Membranes were incubated with primary antibodies diluted in 5% BSA in TBS‐T at 4 °C over night. After washing in TBS‐T, near‐infrared labeled secondary antibodies (IRDye, Li‐Cor Biosciences, Lincoln, NE, USA, dilution 1 : 5000) were applied for 4 h at room temperature. Images were acquired using Azure c600 fluorescent imager.
Cells for protein extraction for western blots shown in Figs 4C and S6 were synchronized in M phase using 5 μm S‐Trityl‐L‐cysteine (Sigma‐Aldrich) and harvested by mitotic shake off before lysis in RIPA buffer.