Ispinesib

Specific inhibitors were used to reveal the possible role of different cytoskeletal elements in mitochondrial transport via TNTs by selectively blocking the polymerisation of microtubules, as well as the ATPase activity of actin- and microtubule-based motor proteins, preventing their binding to the cytoskeletal polymers and thus hindering their normal functioning. Microtubule polymerisation was blocked with 10 or 20 µM of nocodazole (Thermo Fisher Scientific, Waltham, MA, USA), the activity of dynein was blocked with 20 µM of ciliobrevin D (Sigma Aldrich, St. Louis, MO, USA), and kinesin was inhibited with 15 µM of ispinesib (Selleck Chemicals GmbH, Cologne, Germany). The actin-based myosin II motor protein was blocked with 25 µM of para-nitroblebbistatin (Optopharma Ltd., Budapest, Hungary), myosin V with 30 µM of MyoVin-1 (Merk Millipore, Burlington, MA, USA), and the activity of myosin VI was inhibited with 30 µM of 2,4,6-triiodophenol (TIP) (Alfa Aesar, Haverhill, MA, USA). The optimal concentrations for all inhibitors were determined before each experiment. All dyes, inhibitors, or DMSO (Sigma Aldrich, St. Louis, MO, USA) for control experiments were diluted in culture medium.

KIF18A compounds (AM-0277, AM-1882, AM-5308, AM-9022) were synthesized by Amgen. The following compounds were purchased as follows: AM-7710 (Enamine), BI-2536 (Jubilant Biosys), docetaxel (Accord Healthcare), doxorubicin (Sigma-Aldrich), gemcitabine (Zydus Hospira), GF120918 (Sigma-Aldrich), GSK923295 (Selleck), ispinesib (Selleck), nocodazole (Sigma-Aldrich), nutlin 3a (Cayman Chemical), olaparib (AstaTech), paclitaxel (Sigma-Aldrich), palbociclib (Sigma-Aldrich) and vincristine (Tocris).

Ispinesib was obtained from Selleck Chemicals (#S1452). For in vitro experiments, stock solutions of Ispinesib were made in DMSO. Working concentrations were made immediately before use and diluted in cell media. DMSO served as the vehicle control. For in vivo work, working dilutions of Ispinesib were made immediately before use in EtOH followed by Tween-80, and then sterile water at a ratio of 20:25:77.5, respectively. The EtOH, Tween-80, and sterile water mixture served as the vehicle control for in vivo studies.

KPT-185, selinexor (KPT-330), KPT-9058, and KPT-9274 were provided by Karyopharm Therapeutics (Newton, MA). Ispinesib, triptolide, bortezomib, β-nicotinamide mononucleotide, and FK866 were obtained from SelleckChem. All compounds were dissolved in DMSO, except for FK866, which was dissolved in ethanol.