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. 2023 Apr 11;24(6):e56241. doi: 10.15252/embr.202256241

Figure 9. Increased stability of PLK1 may cause mitotic defects in UBAP2L depleted cells.

Figure 9

  • A
    WB analysis of endogenous immunoprecipitation (IP) of IgG or UBAP2L from HeLa cells synchronized in mitosis using 5 μM STLC for 16 h. Proteins MW is indicated in kDa. WB is representative of three independent replicates.
  • B
    WB analysis of endogenous IP of IgG or PLK1 from HeLa cells transfected with control or UBAP2L siRNA for 48 h and synchronized in mitosis using 5 μM STLC for 16 h. Proteins MW is indicated in kDa. WB is representative of three independent replicates.
  • C
    WB analysis of IP under denaturing conditions of WT or UBAP2L KO HeLa cells transfected with plasmids encoding for GFP‐PLK1 and His‐Ubiquitin for 30 h and synchronized in mitosis using 1 μM Paclitaxel for 16 h. The short exposure (s.e.) and long exposure (l.e.) of the membrane blotted against the FK2 antibody that specifically recognizes conjugated but not free ubiquitin are shown. Proteins MW is indicated in kDa. WB is representative of three independent replicates.
  • D
    WB analysis of control (siCTL) or siUBAP2L 48 h‐treated HeLa cells were synchronized with 1 mM monastrol for 16 h, treated with DMSO or with 10 nM of the PLK1 inhibitor BI2536 for 45 min, subsequently washed out from monastrol for the indicated time and collected for protein extraction. This moderate treatment is sufficient to restore the aberrant PLK1 catalytic activity observed in UBAP2L KO cells to the levels of the WT, enabling correct mitotic progression but prevents control cells to progress through mitosis. Proteins MW is indicated in kDa. WB is representative of three independent replicates.
  • E–H
    DAPI staining of the experiment described in (D) showing different mitotic stages (E). Quantification of the percentage of cells with misalignments (F), DNA bridges (G) and micronuclei (H). Scale bar, 5 μm. At least 100 cells from each mitotic stage were quantified for all conditions. Graphs represent the mean of three replicates ± SD (one‐way ANOVA with Sidak's correction *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, non‐significant).

Source data are available online for this figure.