Cells were collected from culture dishes using 0.05% trypsin-EDTA (Gibco), followed by 5-min centrifugation at 300g and resuspended in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS to a concentration of 5 × 106 cells/ml. Twenty microliters of cell suspension was added to the device inlet, generating a pressure gradient for cells to enter the device. The pressure was then balanced by transferring 7 to 8 μl of cell suspension from the inlet to the outlet. Cells were allowed to adhere and spread outside of the channel entrances for 20 to 40 min. All wells of the device were then filled with 120 μl of DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. Devices were incubated at 37°C and 5% CO2 before imaging.

Cells were imaged every 10 min for at least 20 hours on an inverted Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) with automated controls (NIS-Elements, Nikon) and a 10×/0.45 numerical aperture Ph1 objective using time-lapse microscopy. During the experiments, cells were maintained on a stage top incubator (Okolab, Pozzuoli, Italy, or Tokai Hit, Shizuoka-hen, Japan) at 37°C and 5% CO2. For select experiments, cells were imaged using fluorescein isothiocyanate and tetramethyl rhodamine isothiocyanate filters.

Cells were treated with the following pharmacological agents or their corresponding vehicle controls: blebbistatin (50 μM; Sigma-Aldrich), para-nitroblebbistatin (20 μM; Optopharma), colchicine (125 μM; Sigma-Aldrich), CK666 (100 μM; Sigma-Aldrich), LatA (2 μM; Sigma-Aldrich), 2-APB (100 μM; Tocris Biosciences), Bapta-AM (25 μM; Sigma-Aldrich), GsMTx4 (20 μM; Abcam), FTY720 (2 μM; Tocris Biosciences), HC 067047 (5 μM; Tocris Biosciences), ionomycin (500 nM; Sigma-Aldrich), and EGTA (5 mM; Sigma-Aldrich).

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