Fig 2: 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

Fig 3: NEK8-mediated colorectal cancer cell proliferation depended on c-MYC S405 phosphorylation. A Western blots to detect the expression level of c-MYC, cyclin D1, and CDK4 in cancer cells stably expressing Vector + c-MYC WT, NEK8 + c-MYC WT, Vector + c-MYC S405A, NEK8 + c-MYC S405A. B CCK8 assays to detect the proliferation ability of cancer cells from Fig. 7A; n = 3, P(SW48) = 0.0011 or 0.0001, P(Lovo) = 0.0025 or 0.0001. C Detecting the cellular ATP level of cancer cells from Fig. 7A; n = 3, P(SW48) < 0.0001, P(Lovo) < 0.0001. D EdU assays to investigate the DNA replication ability of cancer cells from Fig. 7A; statistical analysis shown; n = 3, P(SW48) < 0.0001, P(Lovo) < 0.0001. E The representative tumor images are shown. F The tumor growth curve is shown; n = 5, P = 0.0103, 0.0072, or 0.0035. G Statistical analysis of tumor weight; n = 5, P = 0.0121, 0.0113, or 0.0024. H Western blots to investigate the expression level of NEK8, c-MYC, and Ki-67 in tumors from Fig. 7E. I The working model is shown. All western blots were conducted three times, and similar results were found; a Student's t-test was applied for statistical analysis

Fig 4: NEK8 overexpression promoted colorectal cancer cell proliferation. A Western blots confirmed the successful construction of SW48 and Lovo stably expressing Vector or NEK8. B CCK8 assays to investigate the proliferation ability of SW48 and Lovo stably expressing Vector or NEK8; n = 3, P(Left) = 0.0009, P(Right) = 0.0025. C, D EdU assays to investigate the DNA replication ability of SW48 and Lovo stably expressing Vector or NEK8; representative images shown (C); statistical analysis shown (D); n = 3, P(SW48) = 0.0047, P(Lovo) = 0.0039. E Detecting the cellular ATP level of SW48 and Lovo stably expressing Vector or NEK8; n = 3, P(SW48) = 0.0007, P(Lovo) = 0.0017. All western blots were conducted three times, and similar results were found; Student's t-test was applied for statistical analysis

Fig 5: NEK8 was upregulated in colorectal cancer. A Assessing the expression level of NEK8 in colorectal cancer tissues and normal colorectal tissues using the GEPIA database. B Analysis of the expression level of NEK8 in colorectal cancer tissues and normal colorectal tissues using TCGA database; P < 0.0001. C Analysis of the expression level of NEK8 in colorectal cancer tissues and adjacent normal colorectal tissues by western blots. D Analysis of the mRNA expression level of NEK8 in colorectal cancer tissues and adjacent normal colorectal tissues by RT-PCR. E–H Analysis of the expression level of NEK8 in colorectal cancer tissues and adjacent normal colorectal tissues by IHC assays; representative images shown (E); statistical analysis of IHC scores grouped by normal and tumor (F), T stage (G), and clinical stage (H); number = 69, P (F) < 0.0001, P (G) = 0.0082, and P (H) < 0.0001. I Analysis of the overall survival time of colorectal cancer patients grouped by NEK8 expression level using the GEPIA database; P = 0.0041. J IHC assays to detect the expression level of NEK8 in colorectal cancer tissues; representative images are shown. K Kaplan–Meier Plotter of colorectal cancer patients grouped by IHC score from Fig. 1 J; P < 0.0001. All western blots were conducted three times, and similar results were found; a Student's t-test was applied for statistical analysis