despite its relative lower expression level. HMN1 lines overexpressing human Bcl-2 were also generated to help dissect the role of Akt in regulating apoptosis (Fig. 3 C). Inhibition of Apoptosis by Constitutively Active Akt and Bcl-2 Both constitutively active Akt and Bcl-2 significantly pro- tected HMN1 cells from apoptosis induced by C2-cer- amide as determined by morphological changes and the extent of nuclear condensation (Fig. 4 A). When treated with 30 mM C2-ceramide for 5 h, most cells overexpressing constitutively active Akt kinase or Bcl-2 remained flat and exhibited normal nuclear morphology, whereas vector- transfected cells showed extensive cell shrinkage, mem- brane blebbing, and nuclear condensation (Fig. 4 A, and data not shown). Quantitation of apoptosis by counting Hoechst-stained condensed nuclei revealed that active Akt and Bcl-2 were approximately equally effective at in- hibiting apoptosis (Fig. 4 B). This is consistent with data showing that C2-ceramide induced DNA fragmentation in the two vector control lines, whereas DNA fragmentation was inhibited in myr-AktD4-129 and Bcl-2–expressing sta- ble cell lines (Fig. 4 C). Overexpressing Constitutively Active Akt Does Not Alter Bcl-2, Bcl-x, or Bax Expression Levels Bcl-2 family proteins play an important role in regulating apoptosis and a growing number of signaling pathways are Figure 6. Bcl-2 but not active Akt blocks cytochrome c release. (A) Active Akt fails to inhibit cytochrome c release induced by C2-cer- amide. Vector control line or two independent active Akt lines were treated with 30 mM C2-ceramide for the indicated period. Cells were then fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cyto- chrome c (top). For the internal control, the blots were stripped and reprobed with antibody to a-tubulin (middle). The cytosolic frac- tion was confirmed to be without mitochondria with an mAb to COX IV (bottom; M represents mitochondria as a positive control for COX IV). Data are from a representative experiment performed three times. Note the delay in cytochrome c release is due to cells be- ing treated with 30 mM C2-ceramide in this experiment rather than 50 mM C2-ceramide as used in experiments shown in Fig. 2 C. (B) Quantification of cytochrome c release. Bars: white, pCR vector; black, myr-Akt clone A7; stippled, myr-Akt clone B10. Data represent mean 6 SEM of three independent experiments. (C) Bcl-2 inhibits cytochrome c release induced by C2-ceramide. Vector control line or two independent Bcl-2 lines were treated with 30 mM C2-ceramide for the indicated period. Cells were then fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cytochrome c (top). For the internal con- trol, the blots were stripped and reprobed with antibody to a-tubulin (middle). The cytosolic fraction was confirmed to be without mito- chondria with an mAb to COX IV (bottom; M represents mitochondria as a positive control for COX IV). Data are from a representa- tive experiment performed three times. (D) Quantification of cytochrome c release. Bars: white, pCMV vector; black, hBcl-2 clone 98-1; stripped, hBcl-2 clone 98-2. Data represent mean 6 SEM of three independent experiments. The Journal of Cell Biology, Volume 151, 2000 488