Orca flash 4

One millilitre of cells in culture medium at a density of 1 × 10⁶/mL–1 × 10⁷/mL was vortexed continuously for 30 s at max speed. Cells were observed, and images were captured on a Zeiss imager Z2 microscope with ORCA Flash4 camera and ×40 oil immersion objective.

MuSCs were plated on a microscopy culture chamber (IBIDI, 80826) and cultured in growth media supplemented as above. The plate was incubated at 37 °C, 5% CO₂ and 3% O₂ in a Pecon incubation chamber. A Zeiss Observer.Z1 connected to a Plan-Apochromat 20x/0.8 M27 objective and Hamamatsu Orca Flash 4 camera piloted with Zen software (Carl Zeiss) was used.

To measure SAC strength in live cells, cells were incubated in a 24‐well plate in full‐growth media in a heated chamber (37°C and 5% CO₂) and imaged with brightfield microscopy using a 10×/0.5 NA objective and a Hamamatsu ORCA‐ER camera at 2 × 2 binning on a Zeiss Axiovert 200M, controlled by Micro‐manager software (open source: https://micro‐manager.org/) or a 20×/0.4 NA air objective and a CMOS Orca flash 4.0 camera at 4 × 4 binning on a Zeiss Axio Observer 7. Mitotic exit was defined by cells flattening down in the presence of nocodazole and MPS1 ± PLK1 or BUB1 inhibitors. MPS1 was inhibited with AZ‐3146 shortly prior to imaging, with or without PLK1 inhibition with BI‐2536 or BUB1 inhibition with BAY‐1816032. In Fig EV2G, cells entering mitosis in the presence of nocodazole were analysed. In Figs 2E, 3H, EV3J and 5A, cells entering in mitosis in the presence of AZ‐3146 were analysed. In Figs 5D and EV5C, cells arrested in mitosis at the time of AZ‐3146 ± BI‐2536 or BAY‐1816032 treatment were analysed.
To measure SAC strength in fixed cells, nocodazole and MG132 ± BI‐2536 or BAY‐1816032 were added first for 30 min to ensure complete inhibition, followed by a time course of AZ‐3146 ± BI‐2536 or BAY‐1816032 in media containing nocodazole and MG132. Cells were fixed and analysed by immunofluorescence, probing for KNL1‐pMELT or BUB1.

WISH, SISH, histological stainings and IHC on sections of all mouse experiments were documented with a Leica DM5000 microscope equipped with a Leica DFC300FX digital camera and the used software was Leica FireCam (Version 1.9.1). Immunofluorescence staining of mouse tissue sections were photographed with the Leica DMI6000 microscope equipped with a Leica DFC350FXR2 camera and the Leica Application Suite X software. Images were processed in Adobe Photoshop CS4 or CC, or FIJI ImageJ version 2.0.0, figures assembled using Adobe Illustrator CS4, CS6 or CC. Xenopus WISH was imaged with a Zeiss SteREO Discovery.V12 or if sections were made with a Zeiss Axioskop 2 mot plus each equipped with an AxioCam HRc in combination with AxioVision 4.7. Fluorescence imaging of Xenopus was performed on a Zeiss AxioObserver with a LSM700 and Zeiss ZEN black software. CBF was documented with a Zeiss Axioskop 2 mot plus equipped with a high-speed Hamamatsu video camera Orca flash 4.0 and Zeiss ZEN blue. CGF analysis was done with a Zeiss Axioskop 2 mot plus combined with a Zeiss AxioCam HSm camera and Zeiss AxioVision 4.7. CBF and CGF was assessed using the ImageJ plugin Particle Tracker^{56 (link)}. All trajectories were further analysed using a custom-made program written in RStudio which calculated the velocity of each particle track^{57 (link)}.