Table 1.
Correlation between FUBP1 expression and clinicopathological characteristics of ccRCC.
Fig 1.
FUBP1 is upregulated in ccRCC tissue and cell lines.
(A) FUBP1 mRNA levels in ccRCC and matched normal renal tissues were analyzed by qRT-PCR. The expression levels of FUBP1 mRNA were normalized to that of PPIA, the internal control. (B) Correlation between FUBP1 mRNA levels and TNM stage of ccRCC. (C) Western blot analysis of FUBP1 protein expression in ccRCC (T) and paired normal tissues (N). (D) Representative immunohistochemistry of ccRCC and corresponding adjacent normal renal tissues. Magnification: 200× or 400×. FUBP1 was localized almost exclusively to the nuclei of cells in ccRCC and matched normal renal tissues. (E) FUBP1 protein levels in ccRCC and adjacent normal renal tissues were determined by IHC assays. (F) Western blot analysis of FUPB1 protein expression in HKC, 786-O, and caki-1 cells indicate that FUBP1 protein levels in ccRCC cell lines are significantly higher than that in the HKC cell line. ****P < 0.0001.
Fig 2.
FUBP1 depletion decreases cell proliferation of 786-O and caki-1.
(A) FUBP1 was knocked down in 786-O and caki-1 cells, and FUBP1 protein levels were monitored by Western blot analysis with the anti-FUPB1 antibody. β-actin was used as the loading control. (B) Assessment of 786-O and caki-1 cell proliferation by MTS assays. (C) Colony formation assays were performed to determine the proliferation of 786-O siRNA1/3, caki-1 siRNA1/3, and the corresponding control cells (786-O-Mock/Scrambled and caki-1-Mock/Scrambled). The area percentage occupied by 786-O-siRNA1/3 and caki-1-siRNA1/3 cells was markedly less than those of 786-O-Mock/Scrambled and caki-1-Mock/Scrambled. (D) Representative images of soft agar colony assay in cells transfected with indicated siRNAs. Original magnification: 100×. Colony count statistics showed a significant reduction in siRNA1/3 786-O and caki-1 cells. Values are expressed as the mean ± SD of three independent experiments, *P < 0.05, **P < 0.01.
Fig 3.
Effects of FUBP1 on cell cycle arrest and levels of cell cycle regulator expression in 786-O and caki-1 cells.
(A and B) Representative data from flow cytometry analysis of the cell cycle distribution in 786-O-siRNA1/3, caki-1-siRNA1/3, and the corresponding control cells (786-O-Mock/Scrambled and caki-1-Mock/Scrambled). The percentage of cells in the G0/G1, S, and G2/M phases was quantified and plotted. (C) Western blot analysis of cyclin D1, CDK4, and CDK6. β-actin was used as the loading control. Values are expressed as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01.
Fig 4.
Effects of FUBP1 on cell apoptosis and expression levels of apoptosis-related proteins in 786-O and caki-1 cells.
(A and B) 786-O and caki-1 cells transfected with FUBP1 siRNAs were stained with a combination of annexin V and PI and analyzed by FACS. Cells positive for annexin V staining were counted as apoptotic cells, and the percentage of apoptotic cells is shown. (C) Western blot analysis showing the levels of caspase-3 and cleaved caspase-3 expression after FUBP1 knockdown in 786-O and caki-1 cells. Values are expressed as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01.
Fig 5.
Levels of c-myc and p21 expression are positively correlated with that of FUBP1 in ccRCC tissues.
The levels of c-myc mRNA (A) and p21 mRNA (B) expression in 56 human ccRCC tissues and the corresponding adjacent normal tissues were analyzed by qRT-PCR. The correlation between c-myc mRNA (C) or p21 mRNA (D) and the FUBP1 mRNA expression in tissues was detected by qRT-PCR. The mRNA levels of FUBP1, c-myc and p21 were showed after FUBP1 knockdown in 786-O (E) and caki-1 (F) cells. Values are expressed as the mean ± SD of at least three independent experiments. *P < 0.05, ** P < 0.01.