Fig 2: Kinase Specificity of HECTD3(A) Western blot of endogenous CRAF in HEK293 cell lysates following 24-hr treatment with AUY922. Substantial degradation is observed in cells transfected with control siRNA, but CRAF levels are stabilized in cells transfected with siRNA against HECTD3. Both immunoreactive bands to the HECTD3 antibody are knocked down by the siRNA; see below. GAPDH levels provide a loading control.(B) Western blots of known HSP90 protein kinase clients in HEK293 lysates and their response to treatment of cells with AUY922. Levels of ErbB2, MASTL, CRAF, LKB1, and CDK4, but not BRAF or PDK1, decrease in response to HSP90 inhibition.(C) MASTL, LKB1, and CRAF degradation following AUY922 treatment is reduced in HEK293 cells transfected with siRNA to HECTD3 compared to a control siRNA. Knockdown of HECTD3 had no clear effect on AUY922-triggered degradation of ErbB2 or CDK4. The panel is a montage of three separate experiments; GAPDH is a loading control for each.(D) Western blot of eYFP-CRAF immunoprecipitated from cell lysates of HEK293 cells treated with increasing doses of AUY922 post-lysis. Ubiquitylation of eYFP-CRAF can be enhanced in HEK293 cell lysates, as well as in intact cells, by the addition of AUY922 in a dose-dependent fashion.

Fig 3: MPS1 is a target of the B55-regulated exit pathway. (A) Unified wiring diagram for the spindle checkpoint and PP2A-B55 pathway. (B–D) Simulation of the system described in the wiring diagram was performed for control (siControl; B), 80% PP2A-B55–depleted (siB55 0.2; C), and 90% MASTL-depleted (siMASTL 0.1; D) conditions. Experimental data points obtained by mass spectrometry are marked by an X, while simulated output is shown in solid lines. (E) Simulation of checkpoint response under conditions used for the checkpoint response assays in Fig. 1 (B and D). (F) Mitotically phosphorylated MPS1-GFP purified from 20 mg of cell lysate was incubated with purified PP2A-B55 or buffer control, and dephosphorylation was analyzed by mass spectrometry. The intensities of the phospho-peptides covering S281 and S821 were plotted.

Fig 4: MASTL depletion significantly impairs cyclin B1 recruitment to the mitotic APC/C.(A) Cyclin B1 binds to the APC/C specifically in mitosis. HeLa cells were synchronised in G2 and M phase by a thymidine block, followed by nocodazole or taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off, and treated for another 2 hours with 5 µM MG132 where indicated. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4. Aliquots of the immunoprecipitates were analysed by western blotting. (B) MASTL silencing impairs the recruitment of cyclin A2 and cyclin B1 to the mitotic APC/C. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4 and aliquots of the immunoprecipitates were analysed by western blotting. Note that equal amounts of APC4 are immunoprecipitated in the mitotic fractions, whereas cyclins A2 and B1 bound to the APC/C are hardly detectable in the MASTL RNAi extracts. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin (right-hand panel). (C) Direct binding of cyclin B1 to the APC/C may depend on extensive APC3 phosphorylation in mitosis. HeLa cells treated as in (B), were subjected to immunoprecipitation using a mouse anti-cyclin B1. G1 phase cells were harvested 90 minutes after treatment with 10 µM of RO-3306. Aliquots of the immunoprecipitates were analysed by western blotting. For cyclin A2 blots in A,B, * denotes an aspecific band that is only observed upon immunoprecipitation.

Fig 5: MASTL depletion increases the frequency of anaphase bridges that may depend on the activity of separase.(A) DNA bridges formed during anaphase are specific to MASTL silencing. Asynchronously growing HeLa cells were transfected with indicated siRNAs, formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using anti-α-Tubulin and DAPI. Arrows indicate DNA bridges connecting the daughter cells in G1 phase. (B) Combined depletion of MASTL and PPP2CA prevents the formation of DNA bridges. HeLa cells stably infected with an shRNA targeting PPP2CA (shPPP2CA) or a control shRNA (shGFP) were transfected with indicated siRNAs. Cells were formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using DAPI. Representative images show a regular cell or two daughter cells that are connected to each other by a DNA bridge (indicated by the red arrow). The amount of cells containing a DNA bridge were scored and quantified for each condition [n = 3; mean±standard deviation (s.d.)]. (C) Western blot analysis to show the effects of indicated shRNAs on total protein levels. A reduction in the protein levels of PP2A-A and PP2A-Cα demonstrates the knockdown efficiency of shPPP2CA. (D) MASTL silencing does not alter the mitotic chromosome morphology. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by a thymidine block followed by subsequent nocodazole treatment for 5 hours. Cells were processed for chromosome spreads 48 hours after transfection (or 24 hours in case of SGOL1 RNAi). Where indicated, cells were treated for 13 hours with 5 µM ICRF-193. Chromosomes were stained using DAPI. The different treatments are categorised based on the mitotic chromosome morphology. Insets demonstrate the morphology of a representative chromosome for each condition. (E) Cyclin B1 and separase physically interact during mitosis. HeLa cells were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 2 hours to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a mouse anti-separase and aliquots of the immunoprecipitates were analysed by western blotting. (F) Absence of MASTL impairs the activation of separase in mitosis. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the separase cleavage product signal was quantified for each condition, starting in mitosis, and corrected for the signal of the same product present in mitotic lysates. The resulting amount was corrected for the total level of separase (full length plus cleavage product). Finally, these values were normalised to 100% at the onset (30 min G1 condition) of mitotic exit (n = 3; mean±s.e.m.). (G) Schematic working model of cyclin B1-mediated separase inhibition. The activation of APC/CCdc20 results in the destruction of cyclin B1 and securin (indicated by the ubiquitin chains), thereby liberating separase from its inhibitors. Subsequently, activated separase triggers the induction of anaphase by cleaving centromeric cohesin (red dots). When cyclin B1 degradation is impaired, its binding to separase will prevent proper sister chromatid separation, leading to the DNA bridges observed in MASTL RNAi cells.