Fig 1: FBL is highly expressed in hepatocellular carcinoma, and high expression of FBL is positively correlated with the occurrence of lung metastasis, indicating a poor prognosis in our cohort. (A) Western blotting analysis of FBL protein expression in hepatocellular carcinoma and adjacent liver tissues. GAPDH was used as an internal reference. In each band, normal tissue is shown on the left, and tumor tissue is shown on the right. (B) RT-qPCR analysis of FBL mRNA expression in hepatocellular carcinoma tissue and adjacent liver tissue. The ordinate is −Δt (the number of PCR cycles of FBL minus the number of PCR cycles of the internal control GAPDH). (C) Representative images of hepatocellular carcinoma paraffin specimens stained by the immunohistochemical method that display the different expression levels of FBL. (D) Comparison of the incidence of lung metastasis in hepatocellular carcinoma patients with high or low expression of FBL. (E, F) Overall survival (OS) and disease-free survival (DFS) between FBL high expression and low expression groups of hepatocellular carcinoma patients in our cohort. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) Student’s t test. (B) paired t-test. (D) χ2 test. (E,F) Log-rank test.
Fig 2: Knockdown of FBL suppresses migration and invasion in HCC cell lines. (A, B) The effect of FBL knockdown on the migration ability of Huh7 and PLC/PRF/5 cells was verified by a scratch wound healing assay. Forty-eight hours after scratching, the differences in the degree of wound healing between the two knockdown FBL experimental groups (siFBL#1 and siFBL#2) and the control group (siNC) transfected with control siRNA were detected. (C) The effect of FBL knockdown on the migration ability of Huh7 and PLC/PRF/5 cells was verified by the Transwell migration assay. Scale bar: 100 µm. (D) The effect of FBL knockdown on the invasion ability of Huh7 and PLC/PRF/5 cells was verified by the Transwell invasion assay. Scale bar: 100 µm. These experiments were performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001. (A–D) random block design analysis.
Fig 3: Knockdown of FBL suppresses hepatocellular carcinoma cell growth in vivo. (A) The knockdown efficiency of FBL knockdown lentivirus (shFBL) on FBL in Huh7 cells was detected by Western blotting analysis. shNC is the control group. (B) Gross tumors excised from the nude mice subcutaneously injected with Huh7 cells stably transfected with shNC or shFBL.. The smallest scale division of the ruler is 0.1 mm. (C) Tumor growth curve chart, tumor sizes in nude mice subcutaneously injected with Huh7 cells stably transfected with shNC or shFBL were measured at days 14, 21, 24, 27, and 30. (D) Weights of gross tumors excised from the nude mice subcutaneously injected with Huh7 cells stably transfected with shNC or shFBL. * p < 0.05, ** p < 0.01, *** p < 0.001. (A,C,D) Student’s t test.
Fig 4: FBL is highly expressed in hepatocellular carcinoma patients, and the prognosis of hepatocellular carcinoma patients with high FBL expression was found to be worse according to data from the TCGA and GEO databases. (A,B) FBL expression in hepatocellular carcinoma and adjacent liver tissues in TCGA; (B) shows a comparative analysis of paired samples. (C,D) FBL expression in hepatocellular carcinoma and adjacent liver tissues in GSE14520; (D) shows a comparative analysis of paired samples. (E,F) Overall survival (OS) and disease-free survival (DFS) between FBL high expression and low expression groups of hepatocellular carcinoma patients in the TCGA. (G,H) Overall survival (OS) and disease-free survival (DFS) between FBL high expression and low expression groups of hepatocellular carcinoma patients in the GSE14520. (A–D) Wilcoxon rank sum test. (E–H) Peto-Peto’s modified survival estimate test.
Fig 5: Knockdown of FBL suppresses proliferation and stemness in HCC cell lines. (A) The protein level of FBL in an immortalized liver cell line (LO2) and HCC cell lines (Huh7 and PLC/PRF/5) were measured by Western blotting. GAPDH was used as a loading control. (B) The knockdown efficiency of two siRNA sequences (siFBL#1 and siFBL#2) on FBL in Huh7 and PLC/PRF/5 cells was detected by Western blot analysis. siNC is the control group. (C) After 48 h post-siRNA transfection, the CCK8 kit was used to detect the effect of FBL knockdown on the proliferation of Huh7 and PLC/PRF/5 cells after 0, 1, 3, and 5 days. The absorbance value at 450 nm detected by the microplate reader was 450 nm OD. (D) After 48 h post-siRNA transfection, the EdU kit was used to detect the effect of FBL knockdown on the DNA synthesis ability of Huh7 and PLC/PRF/5 cells. Scale bar: 50 µm. (E) The effect of FBL knockdown on the stemness of Huh7 and PLC/PRF/5 cells was verified by sphere formation assay. Scale bar: 100 µm. (F) The effect of FBL knockdown on the stemness of Huh7 and PLC/PRF/5 cells was verified by clone formation assay. These experiments were performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) Student’s t test. (B–E) Random block design analysis.
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