Figure 5. Overexpressing constitutive Akt does not change Bcl-2, Bcl-x, or Bax expression in HMN1 cells. Equal amounts of cell lysates from two control vector lines (V1 and V2) or three active Akt lines (A7, B10, and C10) were separated by SDS- PAGE, followed by Western blot with an anti–mouse Bcl-2 antibody, an anti–Bcl-x anti- body, or an anti–Bax anti- body. For the internal con- trol, blots were stripped and reprobed with an mAb spe- cific for a-tubulin. Data are from a representative experi- ment performed three times. Figure 4. Inhibition of apoptosis by constitutively active Akt and Bcl-2. (A) Fluorescence photomicrographs of Hoechst 33342 staining of HMN1 cells transfected with pCMV, human Bcl-2, pCR3.1, or myr-AktD4-129. Cells were treated with 30 mM C2- ceramide for 5 h. (B) Constitutively active Akt and Bcl-2 inhibit ceramide-induced HMN1 cell apoptosis. Cells were treated with 30 mM C2-ceramide for 5 h and apoptosis was assayed by Hoechst Zhou et al. Akt Inhibits Apoptosis after Cytochrome c Release 487 tion of cytochrome c in HMN 1 cells treated with C2-cer- amide. Cell fractionation studies revealed that C2-ceramide treatment leads to cytochrome c redistribution into the cy- tosolic fraction, which can be detected as early as 1 h (Fig. 2 C, top). This observation is consistent with the notion that cytochrome c release occurs before caspase-3 activa- tion. Interestingly, zVAD-fmk did not block cytochrome c release from the mitochondria (Fig. 2 C), even though it inhibited caspase-3 activation and PARP cleavage (Fig. 2, A and B). These results suggest that cytochrome c release from mitochondria during ceramide-induced apoptosis in HMN1 cells is independent of caspase activation. Establishing Stable HMN1 Lines Overexpressing Constitutively Active Akt or Human Bcl-2 One mechanism by which active Akt protects BAF/3 cells from interleukin-3 withdrawal–induced apoptosis is to in- crease the expression level of Bcl-2 (Ahmed et al., 1997). We previously showed that transiently overexpressing constitutively active Akt in HMN1 cells inhibits C2-cer- amide–induced apoptosis; however, the expression levels of Bcl-2, Bcl-x, or Bax were not changed (Zhou et al., 1998; data not shown). The lack of an effect in HMN1 cells may have been due to the low transfection efficiency in our transiently transfected cells or possibly that active Akt may protect neurons from apoptosis via a mechanism dif- ferent from that in BAF/3 cells. To elucidate the cellu- lar mechanisms by which Akt inhibits apoptosis, stable HMN1 lines overexpressing constitutively active Akt were generated. The expression of constitutively active myr- AktD4-129 in these clones was confirmed using anti–Akt antibody (Fig. 3 A) and antiphosphospecific Akt (Ser473) antibody (B). Note that myr-AktD4-129 has a much higher degree of Ser473 phosphorylation than endogenous Akt 33342 staining. Data represent mean 6 SEM for three indepen- dent experiments. (C) Constitutively active Akt and Bcl-2 inhibit DNA fragmentation in HMN1 cells. Cells were treated with 30 mM C2-ceramide for 0, 3, or 5 h, and soluble DNA was extracted for DNA fragmentation assay. Data are from a representative ex- periment performed three times.