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. 2018 May 17;37(14):e96729. doi: 10.15252/embj.201796729

Figure EV4. The under‐loading of CENP‐A to chromatin in DNA2‐null cells is not due to cell cycle changes and is not affected by MMR status.

Figure EV4

  • A, B
    (A) DNA2Flox/+/+ and DNA2Flox/−/− cells were incubated with 1 μM 4‐OHT for 72 h, or 100 ng/ml nocodazole and 10 μM RO‐3306 for 24 h which synchronize cells in G2 phase. Cells were harvested, fixed, stained with propidium iodide, and analyzed by flow cytometry to obtain cell cycle profiles. (B) The quantification of mean ± SD of the cell cycle distribution from three biological repeats.
  • C
    Western blot analysis of chromatin‐bound proteins (after removal of cytoplasmic and nuclear chromatin‐free fractions) extracted from cells treated as described in panel (A).
  • D
    The role of DNA2 in maintaining centromere integrity is not affected by MMR status. MMR‐deficient HCT‐116 and MMR‐proficient HCT‐116‐CH3 cells were transfected with DNA2 siRNAs. Seventy‐two hours after transfection, cells were harvested, and chromatin‐bound proteins (top panel) or whole‐cell lysates (bottom panel) were extracted for Western blot analysis of the indicated proteins. MLH1 (mutL homolog 1) was probed to confirm its expression status in the HCT‐116 cells. CENP‐A was checked as indicator of the centromere integrity.
  • E
    The mean ± SD of the cells in sub‐G1 was from three replicates as in panel (A).

Source data are available online for this figure.