Fig. 2. CUL4-mediated AURKA destruction is independent of known pathways of AURKA degradation.

a RPE1-AURKA-Venus^KI cells treated as in Fig. 1d were scored for mitotic slippage (exit from mitosis in presence of STLC). Percentage of cells undergoing slippage after 3 h treatment with PROTAC-D or Cpd A is shown relative to DMSO-treated cells. Mean values were generated from three identical experiments (where n ≥ 30 cells), plotted with error bars to indicate SDs and tested by ordinary one-way ANOVA with Dunnett’s post-hoc test for significance. b, c Degradation of AURKA-Venus in mitotic-arrested RPE1-AURKA-Venus^KI cells after treatment with PROTAC-D in the presence of APCin/proTAME. Degradation was measured in single cell assays, with data shown as scatter plots (bars to indicate means ± SDs) tested with unpaired t-Test or Mann–Whitney U-test for significance (b) or visualized as a time-course of drug treatment by immunoblot (c). d–f RPE1-Cyclin B1-Venus^KI cells treated with PROTACs to investigate mitotic slippage. Increased degradation of Cyclin B1–Venus following PROTAC-D treatment (d), correlates with increased rate of mitotic exit (e) (both results statistically tested by ordinary one-way ANOVA with Dunnett’s post-hoc test for significance) and is blocked by co-treatment with APC/C inhibitors APCin (40 µM) and proTAME (20 µM) (unpaired t-Test or Mann Whitney U-test for significance) (f). g–i “Non-degradable” AURKA-Venus is degraded efficiently in response to PROTAC-D: AURKA-Venus-Δ32-66 (ΔA-box) expressed in RPE1 cells (tet-inducible RPE1-FRT/TO pool) is sensitive to PROTAC-D (unpaired t-test for significance) (g). WT or Ser51D versions of AURKA-Venus transiently electroporated into U2OS cells are equally sensitive to PROTAC-D treatment in either mitotic cells arrested by STLC (h) or interphase cells (i) (unpaired t-test or Mann–Whitney U-test for significance). Ongoing protein synthesis is likely to mask the degradation rate of Venus-tagged protein in interphase cells.