Fig 1: Downregulation of CREB5 inhibits the invasion and metastasis of CRC cells in vivo and in vitro. a and b Wound-healing assay and transwell migration assay were performed to evaluate the invasive and migratory abilities of CRC cells with different treatments in vitro. c HUVEC tube formation after stimulation with the indicated conditioned medium. d Representative images of the CAM assay. Histograms show the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium. Scale bar: 2 mm. e Orthotopic transplantation with the indicated HCT116 cells in nude mice (n = 9 in each group) was performed, and representative gross images of the livers and intestines are shown. The arrows indicated the tumors. Liver sections were stained with haematoxylin and eosin (H&E). Scale bar: 50 µm. f The Kaplan-Meier method was used to analyse survival curves in the specified treatment groups, and the log-rank test was used to compare differences. ** p < 0.01
Fig 2: Overexpression of CREB5 promotes the invasion and metastasis of CRC cells, but inhibition of MET weakens these effects. a and b The invasive and migratory abilities of CRC cells in vitro with different treatments were evaluated by wound-healing assay and transwell migration assay. c HUVEC tube formation after stimulation with the indicated conditional medium. d Representative images of the CAM assay. Histograms show the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium. Scale bar: 2 mm. e Orthotopic transplantation with the indicated SW480 cells in nude mice (n = 9 in each group) was conducted, and representative gross images of the livers and intestines are shown. The arrows indicate the tumors. Liver sections were stained by H&E. Scale bar: 50 µm. f The Kaplan-Meier method was used to analyse the survival curves of different treatment groups, and the log-rank test was used to compare differences. ** p < 0.01, *** p < 0.001, **** p < 0.0001
Fig 3: CREB5 is upregulated in CRC and associated with a poor prognosis. a and b Real-time PCR and western blotting analysis of CREB5 expression in paired human colon cancer tissues and adjacent noncancerous tissues (p < 0.01). Quantity One software was used to quantify the protein expression levels. c IHC representative images of CREB5 expression in normal intestinal epithelium and CRC tissues. Scale bar: 50 µm. d The paraffin samples of 198 CRC patients were divided into a low-CREB5 expression group (n = 108) and a high-CREB5 expression group (n = 90) based on IHC results. The Kaplan-Meier method was used to analyse survival curves, and the log-rank test was used to compare differences (p = 0.023)
Fig 4: CREB5 regulates MET and binds directly to the MET promoter. a The MET promoter sequence was cloned into pGL3-Basic vector containing the luciferase reporter gene and then transfected into CRC cells with the indicated treatments. b Schematic diagram of the full and truncated MET promoter. c The full-length MET promoter or its truncations were cloned into pGL3-Basic vector containing the luciferase reporter gene and then transfected into HCT116 cells with CREB5 shRNA or empty vector. d ChIP analysis of CREB5 binding to the MET promoter in SW480 cells. ** p < 0.01
Fig 5: CREB5 activates the MET signalling pathway. a GSEA of GSE17538 in MET signalling pathways (ES = 0.43, p < 0.01). b Stable overexpression and interference cell lines were detected by western blotting and real-time PCR. c The expression of MET and downstream signalling molecules in CREB5-knockdown or CREB5-overexpressing cells was observed by western blotting. d CREB5 had an effect on MET by real-time PCR in the indicated cells. e After transient transfection of different amounts of the CREB5-overexpression plasmid in SW480 cells, the protein and mRNA levels of MET were detected by western blotting and real-time PCR, respectively. ** p < 0.01
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