Fig. 4
The endoplasmic reticulum (ER) protein FAM134A is required for rounding pressure during mitosis and timely prometaphase progression. a Co-localization of human FAM134A-EGFP and an ER marker in mitosis. Blue, DNA; Green, FAM134A-EGFP; Red, ER Tracker, an ER marker. Yellow regions represent co-localization between FAM134A and ER. Scale bar, 10 µm. b Relative equilibrium (Rel. eq.) rounding pressure of mitotic and interphase cells upon depletion of FAM134A by RNAi. Values relative to average rounding pressure of control are shown. See Supplementary Fig. 6 for a description of the assay used and for full results for all genes tested. c Depiction of the trans-mitotic height confinement assay used to determine the intracellular pressure of a cell rounding against a wedged cantilever (brown) held at a height h s of 10 µm. P, rounding pressure; F, rounding force. Blue, glass surface. d Representative confocal microscopy images (top) and force traces (bottom) of H2B-mCherry and MYH9-EGFP expressing RNAi treated HeLa cells submitted to the assay depicted in c. Lines represent force traces, open diamonds represent rounding pressure values. NEBD, nuclear envelope breakdown. Scale bars, 10 µm. e Plateau pressure of metaphase cells as depicted in d. f Duration from NEBD to metaphase plate formation (left) and from metaphase plate formation to anaphase onset (right) for cells submitted to the assay depicted in c. Each black diamond represents one cell. ‘(n)’ denotes number of cells measured. Blue bars, mean. Statistical significance was determined by Mann–Whitney U-test (NS, p > 0.05; *p ≤ 0.05; **p < 0.01; ***p < 0.001)
