Fig. 3.

Silencing of Higd-1a expression inhibits cell proliferation. HEK293T and HeLa cells were transiently transfected with scrambled (-) or Higd-1a (H) siRNAs. (A) Total cell extracts were immunoblotted with anti–Higd-1a polyclonal antibody. β-Actin was used as a loading control. (B) Total viable cells were counted by Trypan blue exclusion at the indicated times after transfection. (C) Images of representative cell culture dishes stained with crystal violet were obtained 2 wk after transfection. (D) Flow cytometry cell cycle analysis of HeLa cells 4 d after transfection. (E) Three days after transfection, BrdU was added for an additional 24 h and BrdU incorporation was analyzed by flow cytometry. (F) Annexin V/propidium iodide staining was performed 4 d after transfection. The percentages of annexin V and propidium iodide negative cells (viable cells) were quantified by flow cytometry. (G) Three days after transfection, HeLa cells were stained with rhodamine-123 and relative fluorescence intensity was analyzed by flow cytometry to measure mitochondrial membrane potential. As a positive control for membrane potential changes, cells were treated with 10 μM CCCP for 30 min. Results shown are representative of three independent experiments. Data are means ± SE. **P < 0.01 and ***P < 0.001.