Fig 2: Downregulation of PLZF causes abnormal limb development in chicken embryo Sall4, Plzf or Crbn mRNA expression in E4 chicken embryos was analysed by whole-mount in situ hybridisation. Upper panel shows whole chicken embryo, and lower panel shows right forelimb bud. Sall4, Plzf or Crbn mRNA expression in E4 chicken limb bud was analysed by section in situ hybridisation.Immunoblot analysis of AGIA-GgPLZF in AGIA-GgPLZF expressing DF-1 cells transfected with shControl (shGFP) or shPLZF expression vector.Immunoblot analysis of PLZF from tissue of chicken forelimb or hindlimb bud. Endogenous PLZF protein expression was detected by immunoblot using E4.5 chicken embryos infected with RCAN virus packaging shControl or shPlzf (forelimb shControl [n = 4], forelimb shPLZF [n = 4], hindlimb shControl [n = 2] or hindlimb shPlzf [n = 3]).Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl [n = 3], forelimb shPLZF [n = 5] or hindlimb shPLZF [n = 6]). P-values were calculated by one-way ANOVA with Tukey's post hoc test (**P < 0.01 and ****P < 0.0001).Teratogenic phenotypes of chicken embryos in Fig 6E. Source data are available online for this figure.

Fig 3: Comparison of biotinylation enzymes in biotinylation of neo-substrate.a, b IMiDs-dependent biotinylation assay of neo-substrates by BioID-CRBN in a HEK293T or b IMR32 cells. HEK293T or IMR32 cells stably expressing AGIA-BioID-CRBN were treated with DMSO or 10 µM pomalidomide (Po) in the presence of 10 µM biotin and 5 µM MG132 for 2 h or 6 h. The experiments were repeated twice independently with similar results. c, d IMiDs-dependent biotinylation assay of neo-substrates by AirID-CRBN or TurboID-CRBN in c HEK293T or d IMR32 cells. HEK293T or IMR32 cells stably expressing AGIA-AirID-CRBN or AGIA-TurboID-CRBN were treated with DMSO or 10 µM pomalidomide (Po) in the presence of 10 µM biotin and 5 µM MG132 for 2 h or 6 h. The experiments were repeated twice independently with similar results. e Comparison between AirID-CRBN and TurboID-CRBN of biotinylated proteins detected by LC-MS/MS analysis using IMR32 cells. Overlapping biotinylated proteins were compared between AirID-CRBN and TurboID-CRBN using a Venn diagram. f LC-MS/MS analysis of biotinylated peptides using AirID-CRBN or TurboID-CRBN in IMR32 cells. IMR32 cells stably expressing AGIA-AirID-CRBN or AGIA-TurboID-CRBN were treated with DMSO or 10 µM pomalidomide (Po) in the presence of 10 µM biotin and 5 µM MG132 for 2 h (biological replicates; n = 3). Significant changes in the volcano plots were calculated by Student’s two-sided t-test and the false discovery rate (FDR)-adjusted P-values calculated using Benjamini–Hochberg method are shown in the Supplementary Data 9, 10. a–d Biotinylated proteins were pulled down using streptavidin beads and analysed by immunoblotting. Source data are provided as a Source data file.

Fig 4: AirID-CRBN with thalidomide and IMiDs presents neo-substrate candidates in cells.a Schematic diagram of AlphaScreen-based biochemical assay for detecting IMiDs-dependent interaction between CRBN and neo-substrates. b In vitro interaction assay between CRBN and neo-substrate candidates. The interaction between biotin-labelled bls-CRBN and FLAG-GST-neo-substrate candidates in the presence of DMSO or 20 µM thalidomide (Th), lenalidomide (Le), pomalidomide (Po) or 5-hydroxythalidomide (5HT) was analysed using the AlphaScreen-based biochemical assay. All AlphaScreen signals mean raw luminescent signal in the AlphaScreen-based biochemical assay. Error bars denote the standard deviation (independent experiments; n = 3). c Immunoblot analysis of IMiD-dependent protein degradation of ZNF536, ZNF687 and ZMYM2. HEK293T cells expressing AGIA-ZNF536, Myc-ZNF687 or ZMYM2-AGIA and FLAG-CRBN were treated with DMSO, thalidomide (Th), lenalidomide (Le), pomalidomide (Po) or 5-hydroxythalidomide (5HT) for 16 h. The experiment was repeated three times independently with similar results. d Immunoblot analysis of endogenous ZMYM2 protein levels in IMR32 cells treated with DMSO, thalidomide (Th), lenalidomide (Le), pomalidomide (Po), 5-hydroxythalidomide (5HT) or CC-885 for 24 h. The experiment was repeated three times independently with similar results. e IMR32 and HEK293T cells were treated with DMSO or pomalidomide (Po) for 24 h and ZMYM2 mRNA expression levels were measured by quantitative RT-PCR. Relative mRNA expression was determined using the expression level with DMSO treatment. Error bars denote the standard deviation (biological replicates; n = 4). f Immunoblot analysis of ZMYM2 protein levels in IMR32 and HEK293T cells treated with DMSO or pomalidomide (Po) in the presence of DMSO, 5 µM MG132 or 1 µM MLN4924 for 24 h. g Immunoblot analysis of ZMYM2 protein levels in IMR32 cells treated with DMSO or pomalidomide (Po) for 48 h ZMYM2 band intensity was normalised to tubulin band intensity. All relative band intensities are expressed as fluorescent signals relative to that of DMSO. Error bars denote the standard deviation (biological replicates; n = 3) and P-values were calculated by one-way ANOVA with Tukey’s post-hoc test (*P = 0.031). Source data are provided as a Source data file.

Fig 5: Working ModelIn this working model, we show that volasertib inhibits the kinase activity of PLK1, whereas CC-885 induces both CRBN- and p97-dependent PLK1 ubiquitination and degradation. The combination targets the same oncoprotein PLK1 by different means, providing a reasonable molecular mechanism explanation for this synergistic effect against NSCLC.