Figure 4.

A small percentage of apoptotic cells are detected only after prolonged exposure (>15 h) to 10 μM MG132. (A and B) The effect of MG132 treatment on the expression of annexin V. (A) Untreated HeLa cells or cells that were treated with MG132 for the indicated times were collected, stained with annexin V-FITC and PI, and analyzed by flow cytometry. Formaldehyde treatment for 30 min was used as a positive control for apoptosis. The percentage of total dead or dying cells is indicated on the top of each box (% of cell death) and was defined as the sum of early (lower right box) and late (upper right box) apoptosis. The values were normalized to control, untreated cells. (B) Histogram presenting the results from A as the means ± SEM, from two independent experiments. (C) The cleavage of the apoptotic protein caspase-3 is detected only upon 16 h of exposure to MG132. HeLa cells were treated for the various indicated time points with 10 μM MG132 and then used to prepare total cell extracts. Thirty micrograms from each extract were used for Western blot, and the membrane was probed for caspase-3 and G3BP. The molecular weight (MW) of the caspase-3 cleavage product (Caspase-3-CP) is ∼14 kDa, whereas the precursor is 35 kDa. Shown are representatives of two independent experiments.