Nocodazole

Live cell imaging was used to identify cells at early-prometaphase, late-prometaphase and metaphase. Nocodazole (Thermo Fisher Scientific, Waltham, MA) was added to the culture dish to a final concentration of 32 μM, and dishes were then incubated on the microscope stage for 20 min. After incubation, the temperature was reduced to 25 °C to reduce thermal drift of unattached chromosomes, and full-cell-volume image series with a 250–300 nm z-step and a 100–200 ms exposure time were immediately acquired for the previously staged cells.

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.

hTERT-RPE-1 cells were obtained from ATTC, quarantined and tested for mycoplasma infection. RPE-1 cells were cultured in DMEM F-12+ Glutamax medium (GIBCO) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin (Thermo Fisher) and 0.01 mg ml⁻¹ hygromycin B (Thermo Fisher) at 37°C and 5% CO₂.
For synchronization in mitosis, RPE-1 cells were initially seeded at 17% confluence as this ensured that cells were sub-confluent by the time of nocodazole addition (see below), which we found to be crucial for successful synchronization. After 24 h, palbociclib (PD 0332991, Selleckchem) was added to the medium at a final concentration of 1 μM and cells were incubated for further 18 h. After three washes with phosphate-buffered saline (PBS plus CaCl₂ and MgCl₂) to remove the drug, cells were cultured for 8 h in fresh complete medium before adding 50 ng ml⁻¹ nocodazole (Sigma-Aldrich). After 12 h, cells were harvested by mitotic shake-off, centrifuged at 1000 g for 3 min, washed five times with large volumes of PBS plus CaCl₂ and MgCl₂, and released in fresh complete medium with or without 10 μM MG132 (Sigma-Aldrich) for the appropriate times before collection.
Cells were imaged using a Nikon Eclipse TS100F inverted microscope equipped with a Nikon DS-Fi1 digital color camera with a 5 megapixel sensor.