Figure 5.

NUSAP1 knockdown suppresses the cell growth and increases apoptotic cell death in Huh7 cells. (A) qRT-PCR and Western blot analyses of NUSAP1 in Huh7 cells transfected with siRNA against NUSAP1 or Control siRNA (n = 3 per group). (B) Quantification of colonies from Huh7 cells transfected with NUSAP1 or Control siRNA for 72 h (n = 3 per group) (bar = 1 cm). (C) Levels of cell-cycle proteins (Cyclin D1, Cyclin E1, Cyclin B1, Cyclin A2, p21) were detected in cell lysates of NUSAP1 siRNA transfected Huh7 cells by Western blot with GAPDH as a loading control (n = 3 per group). (D) Huh7 cells were transfected with siNUSAP1 for 72 h and analyzed with flow cytometry by DAPI staining (n = 6 per group). The percentage of cells in the Sub-G1, G₁, S and G2/M phases of the cell cycle are indicated. (E) Huh7 cells were transfected with siNUSAP1 and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G₁, S and G2/M phases of the cell cycle are indicated. (F and G) Representative images and quantification of TUNEL (F) and cl. Casp-3 (G) stainings in Huh7 cells transfected with NUSAP1 siRNA for 72 h (n = 3 per group) (bar = 50 μm). (H) Transwell migration assay for Huh7 cells was determined after transfection with NUSAP1 siRNA or Control (n = 3 per group) (bar = 50 μm). Results are represented as mean ± SEM. ns non-significant, * p<0.05, *** p<0.001, by 2- tailed, unpaired t test.