U2OS, TUBG1-sgRNA-U2OS-TUBG1, and TUBG1-sgRNA-U2OS-TUBG1A429-A432-A435 cells were seeded at a density of 1.5 × 105 cells into 6-well plates. The cells were treated with 6 μM aphidicolin (Biochemica, #10797) for 24 h, followed by pulse-labeling with 25 μM chloro deoxyuridine (Sigma-Aldrich, C6891) for 30 min and sequentially with 250 μM iodo-deoxyuridine (Sigma-Aldrich, I7125) for 30 min. Collected cells were diluted to 5 × 104 cells/mL and then spread and stained as previously described52 (link). Images were acquired with a Zeiss LSM 780 inverted confocal microscope and analyzed with the ImageJ software.
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