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. 2020 Sep 18;39(20):e105505. doi: 10.15252/embj.2020105505

Figure EV1. (related to Fig 1). De novo CENP‐A reloading follows the canonical CENP‐A deposition pathway.

Figure EV1

  1. Image of IAA‐treated cells. IAA escaper is highlighted with a dashed yellow circle, and CENP‐A depleted cells are contoured with red dashed lines. Scale bar, 10 μm.
  2. Schematic for the experiments shown in C.
  3. Quantification of centromeric CENP‐A levels normalized to non‐treated level. Each dot represents one experiment, and error bars represent SD. Unpaired t‐test: *P = 0.0493.
  4. Quantification of the relative number of DLD‐1 (square) and U‐2OS (circle) cells with centromeric CENP‐A at the indicated timing of IAA treatment and recovery. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard deviation (SD) from 3 independent experiments.
  5. Schematic for the experiments shown in F‐H.
  6. Left panel: representative images to confirm M18BP1 knock‐down in late M phase cells. Scale bar, 10 μm. Yellow dashed lines highlight nuclei of daughter cells. Right panel: relative M18BP1 levels in late M/early G1 phase after siRNA knock‐down using M18BP1 antibody. Each dot represents one centromere, and error bars represent standard deviation.
  7. Representative images of de novo CENP‐A reloading upon M18BP1 knock‐down. Nuclei are highlighted with white dashed lines. Scale bar, 5 μm.
  8. Quantification of centromeric CENP‐A intensities in the indicated conditions (relative intensities normalized to CENP‐A level in untreated cells). Each dot represents one experiment (> 30 cells per condition per experiment), and error bars represent SD of 2 independent experiments.
  9. Schematic representation for the experiments shown in J‐K.
  10. Bar graphs showing quantification of centromeric CENP‐A intensities following the indicated treatment. Each dot represents one experiment with at least 30 cells. Error bars represent SD of 2 independent experiments.
  11. Immunoblot of total protein levels in the indicated cell lines and conditions.

Source data are available online for this figure.