Fig. 2: Cyclin B1’s phosphate-binding pocket is critical for mitotic fidelity.

A Experimental outline to synchronize HeLa cells transfected with control (ctrl) or cyclin B1 and B2 siRNAs (siB1&B2) in S-phase via double thymidine block and to induce expression of mNG-cyclin B1^wt/mut by doxycycline (dox) treatment. B Immunoblot assessing depletion of endogenous cyclin B1 and B2 and expression of mNG-cyclin B1^wt/mut. p150 served as loading control. C Quantification of live-cell imaging. The mean of n = 3 or n = 4 independent experiments is shown with standard deviation. Unpaired t-test. **p = 0.002, *p = 0.0149. D Quantification shown in (C) with different y-axis scale for reversine conditions. Unpaired t-test**p = 0.002. E Representative pictures of mitotic mNG-cyclin B1^wt/mut (green) HeLa cells treated with either DMSO or nocodazole and stained for tubulin (red) and DNA (blue). Scale bar = 10 µm. F Timecourse to synchronize cells for STLC washout experiments. Images of representative cells processed at indicated timepoints, 1 to 3 h after STLC washout. Tubulin (green), pericentrin (PCNT) (cyan), CREST (red), and DNA (blue) are shown. Scale bar = 10 µm. G Representative images of siB1&B2 cells expressing mNG-cyclin B1^wt/mut imaged 3.5 h post STLC release. Shown are examples of either lagging chromosomes or chromosome bridges with DNA and CREST in green and red, respectively. Right panel shows the quantification of both phenotypes. The mean with standard deviation of n = 3 independent experiments is shown. Unpaired t-test: **p = 0.0023.