Expression of Ez^WT and Ez^Nter in HS2 and HS1 proerythroblasts. (A) 606HS2 cells were transfected with either pEF-neo Ez^WT-VSVG or pEF-neo Ez^Nter-VSVG or pEF-neo empty vector. Whole-cell lysates from 606HS2-Ez^WT cells, 606HS2-Ez^Nter cells, and 606HS2-neo cells were subjected to Western blotting using anti-VSVG and anti-ezrin antibodies. The membrane was reprobed with an anti–β-actin antibody as a loading control. The apparent molecular weight of endogenous and Ez^WT-VSVG (80 kDa) and the apparent molecular weight of Ez^Nter-VSVG (40 kDa) are indicated on the left of the panel. Fold-increase in ezrin expression in 606HS2-Ez^Nter and 606HS2-Ez^WT was calculated in comparison to 606 HS2-neo cells. Notably, because the anti-ezrin antibody is directed against the C terminal region of ezrin, Ez^Nter could not be detected. (B) Proliferation of 606HS2-Ez^WT, 606HS2-Ez^Nter, and 663HS1-neo cells. Data are means plus or minus SD of 4 independent experiments performed in triplicate. (C) The percentage of dead cells was determined by Trypan blue exclusion assay on 606HS2-Ez^WT cells, 606HS2-Ez^Nter cells, and 606HS1-neo cells at 24 hours and 48 hours of culture. Data are issued from the experiment presented in panel B. (D) Detection of fragmented and condensed nuclei in apoptotic cells by fluorescence microscopy. Representative images after Hoechst staining of 606HS2-Ez^WT cells and 606HS2-Ez^Nter cells cultured for 48 hours. Arrows indicate apoptotic bodies. A total of 3 different fields (500 cells per field) were scored by 2 investigators. Percentage of apoptosis is indicated as means plus or minus SD of 3 independent experiments. Bar corresponds to 15 μm. (E) Processing of caspase-3 in 606HS2-Ez^WT cells, 606HS2-Ez^Nter cells, and 663HS1-neo cells. WCEs were subjected to Western blotting using an anti-cleaved caspase-3 p17. β-actin served as a loading control. (F) 663HS1 cells were transfected with either pEF-neo Ez^WT-VSVG, pEF-neo Ez^Nter-VSVG, or pEF-neo empty vector. Whole-cell lysates were subjected to Western blot using anti-VSVG and anti-ezrin antibodies. The membrane was reprobed with an anti–β-actin antibody as a loading control. Living 663HS2-Ez^WT, 663HS2-Ez^Nter, and 663HS1-neo cells were numbered, and percentages of dead cells were determined by Trypan blue exclusion assay at 24 hours and 48 hours of culture. Data are means plus or minus SD of 3 independent experiments performed in triplicate.