Figure 2.

Akt is necessary for PC-1-mediated resistance to apoptosis. (a) MDCK control (F6) and MDCK^PKD1Zeo (C8/68) were transiently transfected using a mock, a WT (WT-Akt), or a dominant negative (DN_Akt) Akt construct. Apoptosis was induced using TNF-α (see Materials and Methods), and cells were stained with an anti-hemagglutin (HA) antibody (in red) to identify transfected cells, with a transferase-mediated dUTP nick-end labeling (TUNEL) assay (in green) to visualize apoptotic cells and counterstained with Hoechst 33342 (blue nuclei) to visualize all of the cells that were present in the field. Very few apoptotic cells are visible in mock-transfected C8/68 cells, whereas a considerable number are visible in F6 cells that are treated under the same conditions. Similar results were observed when the WT-Akt construct was transfected in these two cell lines, whereas higher rates of HA-positive/TUNEL-positive cells are visible in C8/68 when DN-Akt is transfected in these cells. (b) Same experimental design as in a except that cell lysates were prepared from transiently transfected F6 and C8/68 cells and analyzed by Western blot using a mixture of anti-HA and anti-actin antibodies as a loading control. Equal expression levels were achieved with all constructs in all cell lines. (c) Quantification of the results visualized in a. In mock-transfected cells, the percentage of apoptosis is calculated as the number of TUNEL-positive cells over the number of total cells present in the field. In WT-and DN-Akt-transfected cells, the percentage of apoptosis is expressed as the number of TUNEL-positive cells over the number of HA-positive cells. The ANOVA test was used to perform statistical analysis. The results for each pairwise comparison reached statistical significance (see text for details). (d) Same experiment as in a through c performed in HepG2 cells. Both the percentage of apoptosis and the statistical analysis were performed as in c.