Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK
Figure 5
Bcl-2 and Bcl-xL block the proapoptotic effects of Bcl-xAK.
(A) Subclones of A-375 cells stably transfected with pIRES-Bcl-2 (A375-Bcl-2) or mock-transfected (A375-Mock) were transduced with AdV-AK under OFF or ON conditions. Non-transduced cells (−) were used as additional controls. Numbers of apoptotic cells (sub-G1 cell populations) were determined by flow cytometry after PI staining. (B) SK-Mel-13 melanoma cells were transiently transfected with either Bcl-xL or Bcl-xAK alone or with a combination of both (each 2.5 µg plasmid-DNA). Relative DNA fragmentation values, as determined at 24 h and 48 h after transfection, were calculated with respect to cells that had received only the transfection lipid (white bars). (A, B) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Overexpression of Bcl-2, Bcl-xL and Bcl-xAK, as determined by Western blot analyses, is shown in the insets. (C) The mitochondrial membrane potential (Δψm) was determined by flow cytometry after TMRM staining in A375-Mock and in A375-Bcl-2 at 24 h and 48 h. After transduction with AdV-AK, inducible and non inducible conditions were compared (On/Off). The experiment was performed three times, giving comparable results.