Fig 1: Expression of AGGF1 in CRC tissues and paired normal mucosa. a. Western blot analysis was used to detect AGGF1 protein expression in eight representative paired CRC tissue samples, with GAPDH used as the loading control. b–i: Immunohistochemical staining showed that AGGF1 protein expression was significantly higher in CRC tissues compared with adjacent normal mucosa, with AGGF1 staining mainly observed in the cytoplasm of CRC cells. b, f: Negative AGGF1 staining in normal colorectal epithelium; c, g: Weak AGGF1 staining in well-differentiated CRC; d, h: Intense AGGF1 staining in moderately differentiated CRC; e, i: Strong AGGF1 staining in poorly differentiated CRC; b–e. Original magnification: 50×; f–i. Original magnification: 400 ×
Fig 2: In vitro and in vivo assays. a. he protein level of AGGF1 in one normal colorectal mucosa cell line (FHC) and seven CRC cell lines. The expression level of AGGF1 protein in HCT-116 and RKO cells transfected with AGGF1 overexpression or knockdown vectors was validated using western blotting (b). GAPDH was used to normalize protein level. In vitro, overexpression or knockdown of AGGF1 inhibited or elevated CRC wound healing (c), migration, and invasion (d), respectively, compared with control groups. Upregulation or knockdown of AGGF1 expression facilitated or suppressed the tumor metastasis ability of CRC cells in nude mice. e. Representative images of metastatic tumor colonies in mice (left panel (non-metastasis): the nude mouse without metastatic tumor colonies formed, right panel (metastasis): the nude mouse with metastatic tumor colonies formed). f. HE staining of the metastatic tumor colonies in lung and liver (colored arrows: metastatic tumor colonies). g. The number of metastatic tumor colonies was counted (*p < 0.05, c, f: Original magnification: 100×; d: Original magnification: 200×)
Fig 3: AGGF1 expression in HCC correlates with MVD and prognosis(A), MVD in positive AGGF1 expression patients were significantly higher. (B) and (C), patients with AGGF1 expression had a poorer DFS and OS than those with negative ones.
Fig 4: Representative double immunofluorescence in HCC tissue(A) the AGGF1 positive staining was in the cytoplasm (green) and CD34+ was on the cell surface (red). Sections were counterstained with DAPI (blue). (B and C) are the local magnify of A.
Fig 5: Kaplan–Meier analysis with a log-rank test of survival. Disease-free survival (a) and overall survival (b) were significantly shorter in patients with AGGF1-positive tumors than in those with AGGF1-negative tumors (*p < 0.05 for both, log-rank test). P-values were calculated by log-rank test and p < 0.05 was considered significant
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