After seeding overnight in 35 mm, high µ-dishes (ibidi USA Inc., 81,156), cells were treated as indicated in figure legends, washed twice in PBS and incubated in fixation buffer (3.7% formaldehyde in 100 mM PIPES at pH 6.8, 10 mM EGTA, 1 mM magnesium chloride, and 0.2% Triton X-100) for 10 min at room temperature. Cells were washed as before, treated with Phalloidin-iFluor 488 Reagent (abcam, ab176753) for 1 h in 1–5% BSA in PBS, followed by Hoechst 33342 (Thermo Fisher Scientific, H3570) for 5 min. Mounting medium (Fisher Scientific, P10144) and cover slips were applied onto dried cells. Individually discernible cells were imaged on an Olympus Fluoview 10i. Using ImageJ, the phalloidin channel was thresholded and area and circularity values were measured and integrated into swarmplots using Python’s seaborn library.
For time lapse microscopy, FH-B cells in high µ-dishes were incubated with CellBrite Steady Membrane stain (Biotium, 30108-T) for 30–40 min in the environmental chamber of a Nikon C2 Si microscope at 37 °C and 5% CO2. Treatments were added to the cells through a syringe to reach final concentrations of 4 µM FQI1 or 0.01% DMSO. Immediately thereafter, a time-lapse series was acquired by imaging every 30 s for 30 min at 20× magnification.
For time lapse microscopy, FH-B cells in high µ-dishes were incubated with CellBrite Steady Membrane stain (Biotium, 30108-T) for 30–40 min in the environmental chamber of a Nikon C2 Si microscope at 37 °C and 5% CO2. Treatments were added to the cells through a syringe to reach final concentrations of 4 µM FQI1 or 0.01% DMSO. Immediately thereafter, a time-lapse series was acquired by imaging every 30 s for 30 min at 20× magnification.