Figure 3.

IGFBP4 is a potent tumor suppressor in HCC. (A) Western blot analysis of HepG2 and Huh7 cells transiently transfected with empty vector or IGFBP4-expression plasmid. (B) Cell growth curves of differently transfected HepG2 and Huh7 cells. The growth rates were analysed by MTS assay. The data represent the results of three independent experiments. (C) The colony formation abilities of differently transfected HepG2 and Huh7 cells. The cell foci were visualized by crystal violet staining. All experiments were performed in triplicate. Quantification of cell foci is presented in the bar chart, wherein colony counts are presented as ±S.D. (D) The cell invasion abilities of differently transfected HepG2 and Huh7 cells were assessed by Matrigel transwell assay. Quantification of the cells migrating through the Matrigel-coated filter is presented in the bar chart. (E) The differently transfected HepG2 and Huh7 cells were stained with propidium iodide (PI) and the DNA contents were analyzed by flow cytometry. The cell numbers in individual cell cycles were calculated using ModFit software. The results shown were from three independent experiments. (F) Apoptotic cells were assessed by flow cytometry using PI and Annexin V staining. Cells in the lower left quadrant (PI low, Annexin V low) are viable, those in the lower right quadrant (PI low, Annexin V high) are early apoptotic, those in the upper left quadrant (PI high, Annexin V low) are necrotic, and those in the upper right quadrant (PI high, Annexin V high) are late apoptotic. Data obtained from three separate experiments are expressed as mean ± S.D. (G) Nude mice were subcutaneously injected with 0.5 × 10⁶ HepG2 or Huh7 cells with or without IGFBP4 re-expression. After implantation for 4 days, tumor volumes were measured every 6 days for a period of 28 days (n = 5). Representative images of dissected tumors and tumor volume quantification are shown. *P < 0.05; **P < 0.01; ***P < 0.001.