Plan apochromat 40 1.4 oil dic m27 objective

Slides were imaged using a Plan‐Apochromat 40×/1.4 Oil DIC M27 objective on a Zeiss LSM880 confocal microscope (Carl Zeiss Ltd., Oberkochen, Germany) with Zen software (version 14). For the analysis of interphase cells, large fields of view were acquired and the pericentrin stain was used to identify centrosomes. For the analysis of the patient‐matched Mel270, OMM2.5, and OMM2.3 cells in Figure 4, slides were randomised for blind analysis. Mitotic cells were identified in a methodical manner, scanning a coverslip from right to left, top to bottom, and Z‐stacks covering the depth of mitotic cells were acquired. Zen software was used to create maximum‐intensity projections of the Z‐stacks and images were analysed in ImageJ using the merged pericentrin/centrin channels to identify centrosomes.

A2058 and OCM-1 cells were seeded to coverslip-containing (thickness 1, Assistant, Karl Hecht GmbH & Co. KG, Sondheim/Rhön, Germany) 24-well plates (Sarstedt, Nümbrecht, Germany) one day prior to treatment at a density of 5·× 10⁴ cells/well. Cells were treated with 12.5 and 25 μM of Dau–α-MSH conjugates that were diluted in a serum-free medium for 3 h. Lysosomes were stained with LysoTracker^TM Deep Red (Thermo Fisher Scientific, Waltham, MA, USA, 300 nM for 30 min, followed by nucleus staining with Hoechst 33342 (0.2 μg/mL, 10 min). After washing with phosphate-buffered saline (PBS, Lonza, Basel, Switzerland), cells were fixed with 4% paraformaldehyde for 20 min at 37 °C. Coverslips were mounted to microscopy slides by Mowiol^® 4–88 mounting medium (Sigma-Aldrich, St. Louis, MI, USA). Confocal microscopy images were acquired on a Zeiss LSM 710 (in case of A2058 cells) or Zeiss LSM 780 confocal microscope (in case of OCM-1 cells) (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan-Apochromat 40×/1.4 Oil DIC M27 objective. Hoechst 33342 and daunomycin-conjugates and LysoTracker Deep Red were excited with lasers 405, 488 and 633 nm, respectively. ZEN Lite (Carl Zeiss Microscopy GmbH, Jena, Germany) software was used for image processing.