Fig 1: rs658524G>A mutation reduces CTSW expression via KLF5. (A) Differential expression of CTSW expression between normal epithelial cells and ACGEJ cells. Samples are divided by genotypes. *, ****, and blank represents p-value < 0.05, p-value < 0.0001, and no significance of the Wilcoxon test, respectively. (B) Potential transcription factors of CTSW that might bind to rs658524 are predicted by JASPAR and AnimalTFDB affinity score using DNA fragments containing rs658524G (25 base pairs from the position −84 to −60). (C) JASPAR and HumanTFDB are used to predict the binding site of KLF5 at rs658524. (D) Differential expression of CTSW and KLF5 between normal epithelial cells and ACGEJ cells. (E) pGL3-promoter plasmids are transfected into OE19 24 h after the transfection of siRNA of KLF5. Relative luciferase activity is examined 24 h after the transfection of plasmids with triple repetition. *** represents p-value < 0.001 of the unpaired t-test. (F) EMSA assay of rs658524. Concentration of unlabeled probes is designed to be 20- or 50-fold of biotin-labeled probes. (G) Super-shift EMSA assay of rs658524 using the KLF5 antibody. (H) ChIP-qPCR assay of rs658524 using the KLF5 antibody and rabbit IgG with triple repetition. *** represents p-value < 0.001 of the unpaired t-test. (I) Western blot is performed 48 h after the transfection of siRNA of KLF5.
Fig 2: Flowchart of the study. First, we screened the TCGA-CRC database for differentially expressed genes associated with immunity and metastasis; second, we paired the differentially expressed genes and performed machine learning to fit a prognostic signature containing three gene pairs for prognostic validation in the training set and four independent validation sets; third, we explored the correlation between the prognostic signature and clinicopathological characteristics, immune infiltration, and drug sensitivity; finally, we focused on the role of two genes, FABP4 and CTSW.
Fig 3: The biological function of CTSW in CRC immunity and metastasis. (A) The TCGA-CRC dataset was divided into CTSW-high and CTSW-low expression group based on CTSW expression. GSEA analysis demonstrated that the differentially expressed genes were significantly associated with immunity, EMT, and DNA damage repair pathways; (B) TIMER analysis showed a significant positive correlation between CTSW and CD8+ T cells, CD4+ T cells, and neutrophils in CRC; (C) Western blot assay validated the knockout efficiency of CTSW in HT-29 and HCT-116 cells; (D) Western blot assay probed the expression of EMT-related proteins in CTSW-KO cells; (E) Western blot assay showed the expression of cGAS-STING-related genes in CTSW-KO cells; (F) The migratory capability of CRC cells after CTSW knockout was tested by transwell assay. * p < 0.05; ** p < 0.01; *** p < 0.001.
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