Af7000 widefield fluorescence microscope

Human osteosarcoma cancer cells U2OS transfected with α-tubulin-GFP construct were seeded in two µ- Slide 8 Well (10⁵ cells/well) (ibidi, Gräfelfing, Germany) each. Cells were kept overnight in the incubator at 37 °C and 5% CO₂, then they were treated with different concentrations of crizotinib (0.5 × IC50, IC50, 2 × IC50) or DMSO (negative control) and placed for 1 h on ice. One of the µ-Slide 8 well was stained directly after incubation on ice and the other µ-Slide 8 well was incubated at 37 °C and 5% CO₂ and stained after 24 h. Cells were washed with PBS and then stained with Hoechst 33342 Nuclear Stain (BioVision, Wiesbaden, Germany) at room temperature in the dark for 30 min. Subsequently, cells were washed with PBS to remove excessive Hoechst stain and mounted with Fluoromount-G^®. A AF7000 widefield fluorescence microscope (Leica Microsystems, Wetzlar, Germany) was used to perform the imaging. GFP was detected at 470 nm excitation and 525 nm emission. Hoechst stain was detected at 470 nm excitation and 447 nm emission. Images were analyzed using Fiji ImageJ software [61 (link)].

U2OS human osteosarcoma cells transfected with α-tubulin-GFP construct (30,000 cells/well) were seeded in a µ-Slide 8 Well (ibidi, Gräfelfing, Germany). Cells were placed in a 37 °C/5% CO₂ incubator overnight; then, they were treated with 3 µM (1 × IC₅₀) and 6 µM (2 × IC₅₀) gedunin. DMSO was used as a negative control, vincristine (1 µM) was used as a positive control that inhibits polymerization, and paclitaxel (1 µM) was used as a positive control that inhibits depolymerization. After 24 h of treatment, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. Afterward, the cells were washed twice with PBS, and their nuclei were stained with 4′,6-diamidino-2-phenylindole DAPI (1 µg/mL) (Sigma-Aldrich, Darmstadt, Germany) for 5 min at room temperature. Subsequently, the cells were washed with PBS twice to get rid of any excessive staining. The cells were then mounted with ibidi mounting medium (ibidi) and visualized with an AF7000 widefield fluorescence microscope (Leica Microsystems, Wetzlar, Germany). GFP and DAPI were excited with the blue laser (470 nm). GFP emitted light at 525 nm emission; however, DAPI emitted light at 447 nm. Finally, the fluorescent images were analyzed using Fiji ImageJ software (National Institutes of Health, Bethesda, MD, USA) [42 (link)].

Fixed embryos were embedded in 0.25% agarose to be mounted on a glass bottomed dish. Subsequently, embryos were imaged using a Leica SP5 Multi Photon setup (Leica Microsystems) using a 25× water and 63× glycerol objective followed by a z-stack maximum projection (step size 1 µm).
For generation of time lapse videos, live embryos were embedded in 0.25% agarose to be mounted on a glass bottom dish. Subsequently, embryos were imaged live using a temperature controlled AF7000 Widefield Fluorescence Microscope setup (Leica Microsystems) using a 10× dry objective maintaining the temperature at a constant 28°C. The interval between images was set to 2 min. Computation of colocalization was performed using the 3D colocalization tool in Bitplane Imaris software (V9.3.1).