Cage incubator

Cells were seeded in 96-well plates at 15,000/well and incubated overnight. Then, they were treated with cisplatin or TG as previously described. Images were obtained with an ImageXpress Microconfocal High-Content Imaging System every 5 min for 24 h to record the process of cell death. Live-cell imaging was performed in an Okolab Cage Incubator with a constant temperature of 37 °C with 5% CO₂ and constant humidity. All of the imaging data represent at least three random sites, and the images were processed using the ImageJ software program.

Live RPE1 and HeLa cells were imaged using Bruker Opterra Multipoint Scanning Confocal Microscope⁷⁵ (link) (Bruker Nano Surfaces, Middleton, WI, USA). The system was mounted on a Nikon Ti-E inverted microscope equipped with a Nikon CFI Plan Apo VC ×100/1.4 numerical aperture oil objective (Nikon, Tokyo, Japan). During imaging, cells were maintained at 37 °C in Okolab Cage Incubator (Okolab, Pozzuoli, NA, Italy). A 22 μm slit aperture was used for RPE1 and 60 μm pinhole for HeLa cells. The xy-pixel size was 83 nm. For excitation of GFP and mCherry fluorescence, a 488 and a 561 nm diode laser line was used, respectively. For SiR-dyes, a 640 nm diode laser line was used. The excitation light was separated from the emitted fluorescence by using Opterra Dichroic and Barrier Filter Set 405/488/561/640. Images were captured with an Evolve 512 Delta EMCCD Camera (Photometrics, Tucson, AZ, USA) with no binning performed. To cover the whole metaphase spindle, z-stacks were acquired at 30–60 focal planes separated by 0.5 μm with unidirectional xyz scan mode. The system was controlled with the Prairie View Imaging Software (Bruker Nano Surfaces, Middleton, WI, USA).

Hematopoietic cells are non-adherent cells which are not suitable for live single cell imaging as it is difficult to follow a single cell for certain time point due to their suspension nature and motility. To image a single cell for a specified time period we used specialized Microgrid arrays (MGA-050-02 & MGA-125-02) from microsurfaces Pty Ltd that can hold single cell in individual Microgrid arrays for imaging the same cell for specified time period. After sorting, live polyploid cells were set in the Microgrid array plate following manufacturer specified protocol. Cells were imaged with an in-house Nikon Eclipse Ti-E inverted microscope. The microscope is equipped with automated stage control, a perfect focus, and an Okolab cage incubator to maintain stable humidity, temperature and CO₂ levels. This set up can image live cells for 6-day periods without a reduction in the growth rate or viability of cells [23 (link)]. Images were captured every 20 minutes over a 48-hour period in order to visualize reductive cell division.

For TLFM, appropriate dilutions of cell cultures were transferred to agarose pads containing the appropriate medium on a microscopy slide and covered with a cover glass attached to a 125-μl Gene Frame (Thermo Fisher Scientific) to hold the cover glass on the microscopy slide. TLFM was performed on a Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a ×60 Plan Apo λ oil objective, a TI-CT-E motorized condenser, and a Nikon DS-Qi2 camera. Green fluorescent protein (GFP) was imaged using a quad-edge dichroic (395/470/550/640 nm) and a fluorescein isothiocyanate (FITC) single emission filter. A SpecraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as the light source, using the 470/24 excitation filter. Temperature was controlled with an cage incubator (Okolab, Ottaviano, Italy).
Images were acquired using NIS-Elements software (Nikon), and the resulting pictures were further handled with the open source software ImageJ. The average cellular fluorescence of cells was determined using the open source software Ilastik (55 (link)), which was trained to robustly identify and segment bacterial cells and exclude debris and out-of-focus cells. Background fluorescence was subtracted using NIS-Elements software.

Single U2OS cells stably expressing GFP-tagged G3BP1 (Pelkmans Lab^{64 (link)}) were plated on patterned coverslips following the protocol mentioned above. After spreading for 3–4 h, the cells were switched into media containing 100 nM SiR-tubulin (Spirochrome) and 10 μM verapamil (Spirochrome) and incubated in the dye for at least 4 h. The cells were imaged at 37 °C in imaging media containing Leibovitz’s L-15 medium (ThermoFisher Scientific) with 10% fetal bovine serum, 30 μl ml^–1 Oxyrase (Sigma-Aldrich) and 10 mM lactic acid, with 0.5 mM sodium arsenite (Sigma-Aldrich). Imaging was done on a Nikon Ti2 Eclipse epifluorescent microscope with a cage incubator (Okolab), using ×100 oil objective with numerical aperture (NA) of 1.49 and a Nikon DS-Qi2 camera.

Trans-well migration assays were performed as described previously²² . Trans-well invasion were assessed as trans-well migration assays but membrane was covered with a Matrigel® matrix^{39 (link)}. Briefly, after 24 h, stationary cells were removed from the top side of the membrane, whereas migrated cells in the bottom side of the inserts were fixed, stained, and counted in five different fields (magnification, ×200). At least three independent experiments were each performed in triplicate. Wound healing migration assays were performed from cell monolayer on 6-well culture plate where wounds were made with a sterile 2mm-wide tip. After wash, cells were treated or not with different drugs. Phase-contrast images of the wound were obtained at the time of scratching and after 12 h. Automatic acquisitions were performed on a Nikon microscope (eclipse ti), coupled to a Nikon camera (DS Qi2). The system includes a cage incubator (Okolab, USA) controlling temperature, levels of CO₂ and O₂. Analyses were performed using NIS Element AR software. Brieflly, the analyzes were processed by measuring the area of the injured area to 0 h and 12 h after injury with and without treatment. The injury area before and after the test was measured and compared to the control. The values were plotted as the percentage of the wound closure, with the initial width set to the normalized control.

Lentiviral particles were generated by transfecting HEK293T cells with the lentiviral expression construct with 3rd generation packaging plasmids using TransIT^®-LT1 transfection reagents (Mirus Bio LLC, Madison, WI). The supernatant containing the lentiviral particles was harvested 48-72 h post transfection. HUVECs were transduced with LifeAct-mTurquoise construct (as described in Goedhart et al. 2012 [11 (link)]) cloned into a Lentiviral vector (pLV) overnight, selected with puromycin and grown to confluence on fibronectin-coated Lab-Tek Chambered 1.0 borosilicate coverglass slides (ThermoFisher) in EGM™-2 medium (Lonza). Live cell imaging was done with an inverted NIKON Eclipse Ti equipped with a 60 × 1.49 NA Apo TIRF (oil) objective, perfect focus system, CFP and mCherry filter cubes and an Andor Zyla 4.2 plus sCMOS camera. An Okolab cage incubator and humidified CO₂ gas chamber set to 37 °C and 5% CO₂ were used during imaging. Frames were taken every minute for 4–8 h.