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. 2018 Dec 11;20(1):e46224. doi: 10.15252/embr.201846224

Figure 1. TIAR controls mitotic entry.

Figure 1

  1. HeLa‐H2B/tub cells were transfected with control or TIAR siRNAs for 48 h and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3).
  2. Time from S‐ to M‐phase was measured in siRNA‐transfected HeLa‐H2B/tub cells by time‐lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted).
  3. Western blot analysis of unsynchronized HeLa‐H2B/tub cells was carried out to monitor expression of p(S10)‐H3, total H3, and TIAR in control and TIAR‐depleted cells (mean ± SD; n = 3).
  4. HeLa cells were transfected with control or TIAR siRNAs, alone or together with Cdc25B siRNA. Seventy‐two hours after transfection, p(S10)‐H3‐positive cells were quantified by flow cytometry (mean ± SD; n = 3).
  5. HeLadox‐YFP‐TIARr cells were transfected with control or TIAR siRNAs, and 24 h later, cultured in the absence or presence of 1 μg/ml doxycycline for 48 h. The expression of p(S10)‐H3, total H3, and TIAR was measured by Western blot analysis. The graph shows the quantification of p‐H3 levels normalized to total H3 (mean ± SD, n = 4).
  6. HeLadox‐YFP‐TIARr‐RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4).
  7. HeLadox‐YFP‐TIARr‐dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4).
Data information: Statistical significance was determined by unpaired Student's t‐test; *P < 0.05; **P < 0.01; ***P < 0.001.