Fig. 1

High expression of KCMF1 is associated with poor prognosis of patients with CRC A: mRNA levels of KCMF1 in different types of tumor and normal tissues from the TCGA and GTEx databases. TPM: Transcripts per million; NS: No significance; *: P<0.05, assessed by Student’s t-tests; **: P<0.01, assessed by Student’s t-tests; ***: P<0.001, assessed by Student’s t-tests. B: The correlation between KCMF1 expression and the prognosis of CRC patients in GSE39582 dataset from the GEO database. **: P<0.01, assessed by log-rank tests. C: Representative images of immunohistochemical staining for KCMF1 in CRC tissues and their paired adjacent tissues. D: Immunohistochemical staining scores in the 90 colorectal tumor tissues and their paired adjacent tissues. ***: P<0.001, assessed by Student’s t-tests. E: The survival probability of 90 patients with high or low expression of KCMF1 in colorectal tumors; The KCMF1 scores are ranked by High and Low, with KCMF1-High group (n=45) and KCMF1-Low group (n=45). ***: P<0.001, assessed by log-rank tests. F: The correlation between KCMF1 expression and TNM stage in patients with CRC. *: P<0.05, assessed by Student-Newman-Keuls."

Fig. 2

Effects of KCMF1 knockdown on the proliferation of CRC cells in vitro A: Expression level of KCMF1 protein in HCT116 and HCT15 cells with or without shKCMF1 expression. B, C: The proliferation of HCT116 and HCT15 cells with or without shKCMF1 expression was assessed by MTT assay (B) and colony formation assay (C). ***: P<0.001, assessed by Student’s t-tests. D: Expression levels of the apoptosis-related proteins in HCT116 and HCT15 cells with or without shKCMF1. E: Flow cytometry analysis of the percentage of apoptotic cells in the indicated cells treated with or without Z-VAD-FMK (20 µm) for 48 h. NS: No significance; ***; P<0.001, assessed by Student-Newman-Keuls. F: Expression levels of the cell cycle-related proteins in HCT116 and HCT15 cells with or without shKCMF1. G: Flow cytometry analysis of the cell cycle distribution of HCT116 and HCT15 cells with or without shKCMF1. **: P<0.01, assessed by Student-Newman-Keuls; ***: P<0.001, assessed by Student-Newman-Keuls."

Fig. 3

Effects of KCMF1 knockdown on the signaling pathways in human CRC cells A: Volcano plots show differentially expressed genes affected by KCMF1 knockdown in HCT116 cells. The significant different genes are determined with the following criteria: |Log2 FC|>1 and P<0.05. FC: Fold change. B: GO analysis of the down-regulated genes in HCT116 cells with KCMF1 knockdown. P<0.05 and Log2 FC≤-1 were used as the criteria to define down-regulated genes. C: Enrichment analysis of the KCMF1-regulated genes with HALLMARK gene signatures, the significantly enriched signatures were ranked by P value, the bar plot shows the top 10 signatures. D: GSEA of the transcriptional profiles of KCMF1-koncokdown HCT116 cells and control cells with the TNFα-NF-κB signature. E: Spearman correlation analysis was performed between BIOCARTA_NFKB_PATHWAY and KCMF1 expression based on TCGA database cohort. F: The TCGA cohort were classified into four groups by both KCMF1 expression and NF-κB activity and subjected to Kaplan-Meier overall survival analysis. The activity of NF-κB signaling was determined by the GSVA score of the gene set BIOCARTA_NFKB_SIGNALING. **: P<0.01, assessed by log-rank tests."

Fig. 4

Knockdown of KCMF1 inhibits the activation of the NF-κB signaling in human CRC cells A: mRNA levels of KCMF1, BCL-XL, XIAP and CIAP in HCT116 and HCT15 cells with or without KCMF1 knockdown. *: P<0.05, assessed by Student-Newman-Keuls; **: P<0.01, assessed by Student-Newman-Keuls; ***: P<0.001, assessed by Student-Newman-Keuls. B: Expression of the phosphorylated proteins related to NF-κB signaling in HCT116 and HCT15 cells with or without KCMF1 knockdown. C: Activity of the NF-κB-reporter in HCT116 and HCT15 cells with KCMF1 knockdown. **: P<0.01, assessed by Student-Newman-Keuls; ***: P<0.001, assessed by Student-Newman-Keuls. D: Immunofluorescence of p65 (green) in HCT116 and HCT15 cells treated with TNFα (10 ng/mL) for 5 min after KCMF1 knockdown. DAPI stains nuclear DNA (blue). Scale=20 microns. E: Quantification of p65 nuclear and cytosolic localization was assessed in the indicated cells, as shown by a ratio of nuclear to cytoplasmic fluorescence. **: P<0.01, assessed by Student-Newman-Keuls. F: p65 cellular localization in HCT116 and HCT15 cells with or without KCMF1 knockdown."