Fig 2: Wnt signaling induces CRBN-mediated degradation of a subset of endogenous substrates.a HEK cells were transfected with the indicated smart-pool siRNA, followed by PBS or Wnt3a treatment for 24 h. Extracts of these cells were evaluated by immunoblotting. A representative immunoblot (n = 3 independent experiments) is shown. b–d HEK cells were co-treated with cycloheximide along with PBS or Wnt3a. Extracts of these cells were evaluated by immunoblotting. CK1a levels from immunoblots were quantitated, normalized to HSP90 levels, and plotted to determine the turnover of the indicated protein in response to Wnt3a (mean ± SEM, n = 3 independent experiments). e–g HEK cells were transfected with control (CTRL) or one of two distinct CRBN siRNA and co-treated with Wnt3a and cycloheximide. Extracts of these cells were evaluated by immunoblotting. CK1a levels from immunoblots were quantitated, normalized to HSP90 levels, and plotted to determine the turnover of the indicated protein in response to Wnt3a (mean ± SEM, n = 3 independent experiments). Asterisks in b–g indicate statistical significance (two-way ANOVA analysis, ***p value < 0.001, ****p value < 0.0001). h Extracts of HEK cells treated for 24 h with different doses of Wnt3a or lenalidomide were evaluated by immunoblotting. A representative immunoblot (n = 3 independent experiments) is shown.

Fig 3: CRBN modulates Wnt function in zebrafish.a A schematic of transverse views of a zebrafish head indicating phenotypes regulated by Wnt signaling. White arrows indicate eyes and the balbis indicates the distance between eyes. b The transverse views of a (i) WT zebrafish head, a WT zebrafish head injected with (ii) crbn mRNA, or (iii–v) one of three distinct crbn guide RNAs (sgRNA) along with dCas9 mRNA, in the absence or presence of (iv) crbn mRNA or (v) ß-catenin (ctnnb1) mRNA. White arrows indicate eyes, the gray arrow indicates merged eyes, and the balbis indicates the distance between eyes. Scale bar = 200 µm. c, e and g Quantifications of the indicated eye phenotypes are shown. Asterisks indicate statistical significance (Fisher’s exact test, *p value < 0.05, ****p value < 0.0001). d and f RNA extracts from the indicated zebrafish (20 hpf) were used to determine the expression of three Wnt target genes using quantitative RT-PCR. Quantification of gene expression in indicated zebrafish embryos (mean ± SEM, n = 3 independent pools of zebrafish embryo) is shown. Asterisks indicate statistical significance (two-tailed Student’s t-test, *p value < 0.05, ***p value < 0.001, ****p value < 0.0001). lef1 lymphoid enhancer-binding factor 1.

Fig 4: CRBN, but Not GSPT1, Mediated the Synergistic Effect of Volasertib and CC-885(A) Cell cycle distribution of A549 CRBN-/- cells treated with 50 nM volasertib, 50 nM CC-885, or both for 24 h. (B) Flow cytometry demonstrated the effects of 50 nM volasertib and the combination of 10 or 50 nM CC-885 with 50 nM volasertib on A549 CRBN-/- cells for 48 h. Error bars indicate the means ± SD. (C) A549 CRBN-/- cells were cultured in a 96-well plate treated with indicated doses of volasertib for 24 h, and CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. (D) Morphological changes of the indicated doses of volasertib on A549 CRBN-/- cells for 24 h. Cells were imaged using a phase-contrast microscope. (E) A549 cells were infected with lentiviral vectors expressing control shRNA or any of the three GSPT1-specific shRNAs. Four days after infection, cells were collected for immunoblot analysis with the indicated antibodies. (F) A549 cells infected with lentiviral vectors expressing control shRNA or any of the three GSPT1-specific shRNAs for 4 days were cultured in a 96-well plate and treated with either DMSO or 50 nM volasertib for 24 h, and CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. (G) A549 cells were treated with 50 nM volasertib, 50 nM CC-885, or both for 24 h. Cells were harvested and subjected to western blot with the indicated antibodies. (H) A549 cells were transfected with Flag-GSPT1 WT or MUT (G575N) for 36 h, and 1µM CC-885 was added for an additional 8 h. Cells were then harvested and subjected to western blot with indicated antibodies. (I) A549 cells transfected with Flag-GSPT1 WT or Flag-GSPT1 MUT (G575N) for 36 h were cultured in a 96-well plate and treated with either DMSO or 50 nM volasertib for 24 h, and CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. ***p < 0.001.

Fig 5: Cleavage of CRBN at Asp9 (D9) reduces its stability. (A) CRBN is cleaved at the N-terminus upon CASP-8 activation. HeLa cells were transfected with CRBN-Flag plasmid for 48 h and treated with DMSO or TRAIL (100 ng/ml) and Btz (0.5 µM) for 24 h. The resulting cell lysates were subjected to immunoblotting analysis. (B) Amino acid sequence alignment for CRBN from human, mouse, rat, and western clawed frog (Xentr). Conserved Asp (D) residues were indicated in red. (C) CRBN is cleaved at Asp9 (D9) upon CASP-8 activation. HeLa cells were transfected with the WT and CRBN Asp (D) to Ala (A) mutants for 48 h and then treated with DMSO or TRAIL (100 ng/ml) and Btz (0.5 µM) for 24 h. Cell lysates were used for immunoblotting. (D) Deletion of the N-terminal nine amino acids in CRBN reduces it stability. WT CRBN and Del9-CRBN mutants were expressed in HEK293T cells and split to 24-well plates. At 48 h post-transfection, cells were further treated with cycloheximide (CHX, 100 µM) for the indicated time. The cell lysates were immunoblotted with the indicated antibodies. Mean ± SEMs were from three independent biological replicates. Two-way ANOVA, **P < 0.01; Clvd, cleaved.