Fig 1: Design and characterization of different E3 ubiquitin ligase-based AURKA PROTACs. A) Crystal structure of AURKA in complex with MLN8054 (PDB code 2×81). The lysine residues on the surface of AURKA are highlighted. B) Chemical structures of AURKA PROTAC based on MLN8237 and the ligands of CRBN, VHL, and cIAP. MW, molecular weight (Daltons). C) SPR sensorgrams and KD values of MLN8237 and AURKA PROTACs binding to the AURKA protein. D) Dual-colored fluorescence SPPIER (DF-SPPIER) imaging of ternary complex formation in HEK293T cells with PROTACs and E3 ligands induction (500 × 10-9 m) for 3 h. Fluorescence histogram of the line across the cells (normalized fluo., normalized fluorescence intensity) and the log10 normalized sum of pixel fluorescence intensity (normalized SPPIER, log10 (intensity + 1)) of yellow droplets in each cell are shown on the right. Scale bar: 5 µm. E) Degradation of endogenous AURKA in KG1A cells following 6 h treatment with the indicated concentration of PROTACs. F) Degradation of endogenous AURKA in KG1A cells following 6 h treatment with the indicated PROTACs (500 × 10-9 m) and E3 ligase ligands. G) Degradation of endogenous AURKA in KG1A cells with or without doxycycline (DOX, 0.5 µg mL-1) treatment for 72 h following 6 h treatment with the indicated PROTACs (500 × 10-9 m). H) TMT-based quantitative proteomics after treatment with dAurA383 (500 × 10-9 m), dAurA450 (500 × 10-9 m) or the DMSO Vehicle for 6 h in KG1A cells. The differentially expressed proteins are presented in the volcano plot. I. KG1A cells were synchronized at the G1/S boundary by a double thymidine method, released into fresh media, and harvested at the indicated times (T/T release). The cell cycle profile was assayed by FACS with propidium iodide (PI) staining. Ctrl, proliferating KG1A cells under normal cell culture condition were used as control. J) The protein expression levels of the indicated proteins after T/T release were measured by Western blot. K) Schematic depiction of the relative protein levels of AURKA, CRBN, cIAP1, and VHL in KG1A cells. Statistics, significance: one-way ANOVA with Bonferroni correction (D); ns, not significant; ***P < 0.001.