Fig 1: CKAP2 expression level is boosted in CRC.(A) Analyzing CKAP2 expression in CRC using the GEPIA database (TCGA-COAD and READ). (B) Analyzing CKAP2 expression level in 5 pairs of CRC tissues and adjacent tissues in the GEO database GSE143939. (C) Detection of CKAP2 mRNA expression level in CRC cell lines and a normal human colon epithelial cell line (FHC) by qRT-PCR. (D) Detection of CKAP2 protein expression in CRC cell lines and FHC cells by western blotting. ***p < 0.001, **p < 0.01, *p < 0.05. We executed each experiment three times.
Fig 2: CKAP2 facilitates the proliferation of HUVECs and angiogenesis by influencing the tumor microenvironment.(A) Protein expression levels of CKAP2 and CD31 (microvascular marker) were stained in CRC tissues by immunofluorescence. Cancer/THP-1 co CM was constructed by co-culturing HCT116/SW480 cells transfected with pcDNA3.1-NC/pcDNA3.1-CKAP2 and THP-1 cells in a 6-well plate for just 48 h, centrifuging to eliminate debris, and HCT116/SW480/THP-1 co CM was then obtained. The HCT116/SW480/THP-1 co CM was used to culture HUVECs. (B) CCK-8 assay was used to detect the proliferation of HUVECs. (C) Tube formation assay could detect the angiogenesis of HUVECs. ***p < 0.001, **p < 0.01, *p < 0.05. We executed each experiment three times.
Fig 3: TFDP1 directly regulates CKAP2 expression.(A) Online prediction of binding sites between TFDP1 and CKAP2 promoter sequences through JASPAR (https://jaspar.genereg.net/). (B, C) The interaction between TFDP1 and CKAP2 in HCT116 and SW480 cells was confirmed using dual luciferase reporter assay and co-immunoprecipitation assay. (D) GEPIA online analysis of the correlation between the TFDP1 and CKAP2 expression levels in COAD (Colon adenocarcinoma) and READ (Rectum adenocarcinoma) databases. (E) Western blotting could detect protein levels of TFDP1 and CKAP2 in HCT116 and SW480 cells. (F) Western blotting could detect protein levels of TFDP1 and CKAP2 in HCT116 and SW480 cells. *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was executed in triplicate.
Fig 4: Overexpression of CKAP2 partially reverses the effect of TFDP1 downregulation on CRC cells.(A) The CKAP2 protein level in different groups (si-NC, si-TFDP1, si-TFDP1+pcDNA-CKAP2) of HCT116 and SW480 cells was detected by western blotting. (B) The proliferation of HCT116 and SW480 cells was assessed by EdU assay. (C) Transwell assay was conducted to detect the migration ability of HCT116 and SW480 cells. (D) Transwell assay was conducted to detect the invasion ability of HCT116 and SW480 cells. *p < 0.05, **p < 0.01, ***p < 0.001. #p < 0.05, ##p < 0.01, ###p < 0.001. Each experiment was executed in triplicate.
Fig 5: CKAP2 downregulation inhibits in vivo tumor growth and metastasis.(A) HCT116 cells were inoculated in nude mice, and the size of the subcutaneous tumor was measured every 7 days during the 7-35 day period. (B, C) Tumor volume and tumor weight were displayed. (D) Positive staining of Ki-67, CKAP2, CD163, and CD31 in tumor tissues was detected by IHC staining. (E) The IVIS was used to monitor tumor lung metastasis in mice. (F) The lung tissues of nude mice were used for HE staining to observe lung metastasis of CRC. **p < 0.01.
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