Fig. 6: KDM3A regulates Wnt/β-catenin signaling at the post-translational level.

A Representative western blot images show the nuclear and cytoplasmic expression of KDM3A in NSPCs. The experiments were repeated at least three times. B Western blotting reveals that the expression levels of p-β-catenin and CK1α are significantly increased in KO NSPCs. The experiments were repeated at least three times, and quantification data are presented as mean ± SEM. Statistical significance was determined by Student’s t test (indicated as *, **, and *** for p < 0.05, 0.01, and 0.001, respectively). C Overexpression of Kdm3a or Kdm3a(H1122A) decreases the expression level of CK1α, as shown by western blotting. The experiments were repeated at least three times, with quantification data represented as mean ± SEM. Statistical significance was determined by Student’s t test (indicated as *, ** for p < 0.05, 0.01, respectively). D Western blotting demonstrates that the degradation rate of CK1α is significantly lower in KO NSPCs compared to WT NSPCs after treatment with 20 µg/mL cycloheximide. Additionally, CK1α accumulation is significantly higher in KO NSPCs than in WT NSPCs with 10 µM MG-132 treatment. The experiments were repeated at least three times, and quantification data are represented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test (indicated as *, **, and *** for p < 0.05, 0.01, and 0.001, respectively). E Immunofluorescent images show that KDM3A colocalizes with CK1α in the cytoplasm. The experiments were repeated at least three times. F HEK-293 FT cells transfected with KDM3A-Flag and CK1α-V5 demonstrate interaction between exogenous KDM3A and CK1α, detected by reciprocal Co-IP assay. The bands of CK1α-V5 and KDM3A-Flag are indicated by the arrowheads. The experiments were repeated three times. G Co-IP assays indicate that endogenous KDM3A physically interacts with CK1α. Note that c-terminus depletion (KO) does not affect the interaction between KDM3A and β-catenin but does affect the interaction between KDM3A and CK1α. The experiments were repeated three times. H Ubiquitination assays demonstrate levels of ubiquitinated CK1α in WT and KO NSPCs following transfection of CK1α-V5 and Ub-HA. The experiments were repeated five times. I HEK-293 FT cells were transfected with KDM3A-EGFP, CRBN-Flag, and CK1α-V5; interaction between KDM3A, CRBN, and CK1α was detected by reciprocal Co-IP assay. The experiments were repeated three times. J Representative western blot images show that lenalidomide reduces the elevated expression of CK1α in Kdm3a siRNA-treated cells. The experiments were repeated three times.