Monastrol

The human hTERT RPE-1 epithelial cell line immortalised with hTERT (kind gift of Prof. Jonathon Pines) and the derived stable cell lines described above were grown at 37 °C and 5% CO₂ in complete DMEM/F12 (Dulbecco’s Modified Eagle Medium F-12) supplemented with 10% tetracycline-free foetal bovine serum (FBS).
When indicated, cells were treated as follows: (a) 100 μM monastrol (Tocris, Bristol, UK) for 12 h to arrest cells in prometaphase; (b) 6 μM RO-3306 (Sigma-Aldrich) for 22 h to block entry into mitosis; (c) 0.5% FBS for 42 h to induce quiescence; (d) 2 mM thymidine (Sigma-Aldrich) for 24 h followed by 10 h release in thymidine-free medium and mechanic shake off, to collect and re-plate mitotic cells; (e) for Brefeldin A treatment (200 ng/mL; Sigma-Aldrich), cultures were presynchronised as in (d) and treated 10 h after thymidine release, for the following 12 h; (f) for cytochalasin B (cyto B) treatment (6,25 μM; Sigma-Aldrich), asynchronous cultures were treated for 24 h, 1 h after dox induction. Mock-treated cultures were incubated with 0.1% DMSO (a−b and e−f) or DMEM/F12 supplemented with 10% FBS (c–d).
For MT depolymerisation experiments, cells were incubated on ice in the presence of 10 μM nocodazole (Sigma-Aldrich) while control cultures were kept at 37 °C in 0.1% DMSO; after 15 min cultures were fixed and processed for immunofluorescence (IF) as indicated.

HeLa cells were cultured in Dulbecco's modified Eagle's Media (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, penicillin and streptomycin). For inhibition studies, cells were treated with 10 µM monastrol (1305, Tocris) for Eg5 inhibition for 3 h or 200 nM NMS-P715 (475949-5MG, Merck) for MPS1 inhibition prior to filming or fixation. 2ME2 (Sigma) was added directly to DMEM containing FCS (Invitrogen). Cells were transfected with siRNAs as described [30 (link)]. All siRNA oligos used against TAO1 are listed in the electronic supplementary material. For Control-St siRNA treatment, negative control (12935-300, Invitrogen) was used. All siRNA transfections were performed using Oligofectamine (Life Technologies) according to the manufacturer's instructions. For tetracycline induction, cells were treated with 1 ng ml^–1 tetracycline (Sigma).

Human osteosarcoma U2OS, colorectal carcinoma HCT116 and breast adenocarcinoma MDA-MB-231 cancer cell lines were purchased from American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium-high glucose (Gibco), 10% FBS (GemCell) and 100 units/mL penicillin. Chemicals used were as follows: 100 ng/μL Nocodazole (N3000) (US Biological), 150 nM Paclitaxel (Taxol) (P1792A) (US Biological), 3 mM Thymidine (T5290) (Sigma), 100 μM Monastrol (1305) and 2 μM ZM447439 (2458) (TOCRIS). Lipid biosynthesis inhibitors used were as follows: Triascin C (ab141888) (Abcam), TOFA (T6575), C75 (C5490) and Orlistat (O4139) (Sigma). For cell synchronisation, cells were subjected to 24 h of serum starvation (0% FBS) followed by 3 mM thymidine arrest (G1/S arrest). Cells were released from thymidine for 3 h before the addition of fresh media with respective drugs.

Log-phase cells (6 × 10³ per well) were seeded into 96-well plates. Following 24 h incubation, media were replaced with media containing paclitaxel, cisplatin, Monastrol, tunicamycin, SP600125, KIRA6 or GSK2656157. Monastrol and SP600125 were obtained from Tocris Bioscience (Bristol, England, UK) and tunicamycin from Sigma-Aldrich (St. Louis, MO, USA), KIRA6 from Calbiochem (San Diego, CA, USA) and GSK2656157 from Selleckchem (Houston, TX, USA). Cells were quantified by sulforhodamine B (SRB) assay.

The human U2OS osteosarcoma cell line (ATCC: HTB-96) was grown at 37°C in a 5% CO₂ atmosphere in DMEM, supplemented with 10% fetal bovine serum. For synchronization, cells were subjected to a 24 hours block in 2 mM thymidine. Cultures were then released from the G1/S arrest by washing away the thymidine and adding fresh medium containing 30 μM deoxycytidine; 6 hours post-release cultures were treated with the C20 or C23 compounds at the indicated concentration. Mock-treated cultures were incubated with DMSO. After further 4 hours cells were harvested and analyzed. When indicated, monastrol (100 μM, TOCRIS, cat. 1305) was added 2 hours before fixation.