Fig. 4

NEK8 positively activated the MYC signaling pathway by regulating the poly-ubiquitin level of c-MYC. A GSEA analysis of GSE17537 dataset; P < 0.0001, FDR < 0.0001. B Western blots to detect the expression level of identified proteins of SW48 and Lovo stably expressing Vector or NEK8. C Western blots to detect the expression level of identified proteins of SW48 and Lovo stably expressing sgNC or sgNEK8. D Transfecting HA-NEK8 WT and HA-NEK8 enzyme deficient mutants (NEK8-FL-KD and NEK8-FL-jck) into SW48 and Lovo cells; Western blots to detect the expression level of identified protein. E Western blots to detect the expression level of identified protein of SW48 treated by DMSO, CQ (20 uM), or MG132 (5 uM). F Co-transfecting his-ub plasmid and Vector or HA-NEK8 plasmid into SW48 and Lovo cells; MG132 to inhibit endogenous c-MYC degradation; co-IP and western blot to investigate the poly-ubiquitin level of c-MYC. G Transfecting his-ub plasmid into sgNC or sgNEK8 cancer cells; MG132 to inhibit endogenous c-MYC degradation; co-IP and western blot to investigate the poly-ubiquitin level of c-MYC. H Cycloheximide (CHX, 50 uM) was applied to SW48 cells stably expressing sgNC or sgNEK8 at the indicated time; western blots to detect the degradation rate of c-MYC. I Statistical analysis for Fig. 4H; n = 3, P = 0.0007 or = 0.0009. J Statistical analysis for Figure S3D; Tested by Pearson's correlation test, n = 69, P < 0.0001. K. IHC score greater than 6 was considered high; otherwise, it was considered low; Statistical analysis for Figure S3D; Tested by Chi-Squared Test, P < 0.0001. L Statistical analysis for Figure S3E; Tested by Pearson's correlation test, n = 14, P = 0.0097. All western blots were conducted three times, and similar results were found