Regulation of Bcl-2 Proteins Independent of BH3 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 (2) 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 mg 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 (Dym) 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. doi:10.1371/journal.pone.0034549.g005 Bax were seen in mitochondrial extracts. In this assay however, Bax translocation and activation is underestimated as some cytosolic contaminations (up to 5%) were still left in mitochondrial fractions seen by the cytosolic marker GAPDH. This may explain the weaker bands of Bax already before induction of Bcl-xAK expression (Fig. 6B). The localization of Bcl-xAK itself appeared as an important step. When comparing 24 h with 48 h, the amount of Bcl-xAK in the cytosol significantly decreased at 48 h by 2–3-fold in all three cell lines. Equal loading of cytosolic extracts was proven by b-actin (Fig. 6A). The direct comparison of the mitochondrial extracts at 24 h and 48 h clearly showed almost no Bcl-xAK in Mel-2a and only weak bands in the two A-375 clones at 24 h. The mitochondrial localization of Bcl-xAK however strongly increased at 48 h (Fig. 6B). Simultaneous decrease of Bcl-xAK in the cytosol and its strong increase in mitochondria at 48 h clearly proved mitochondrial translocation of Bcl-xAK, which is suggestive as a critical step for induction of apoptosis. Importantly, the mitochondrial translocation of Bcl-xAK was not prevented by Bcl-2, whereas cytochrome c release and Bax translocation were completely blocked (Fig. 6A; B). No interaction of Bcl-xAK with other Bcl-2 family members For investigating whether Bcl-xAK might directly interact with other Bcl-2 proteins, SK-Mel-13 melanoma cells were transiently transfected with myc-tagged copies of Bcl-xAK, Bcl-xL or Bax. Following immunoprecipitation with anti-Myc microbeads, bind- ing of Bcl-2, Bax, Bad, Noxa and Puma was investigated by Western blotting. Mock transfected cells were used as controls and ruled out non-specific precipitations by the microbeads. On the other hand, Myc-tagged proteins were efficiently immunoprecip- itated, as seen in the pellet (P) fractions after incubation with the Myc antibody (Fig. 7A, panels 1–3). The binding analyses revealed characteristic interactions, thus proving the reliability of the assay. Thus binding of Bcl-2 to myc- Bax, binding of Bax to myc-Bcl-xL and myc-Bax as well as binding of Bad to myc-Bcl-xL were seen (Fig. 7A). Apoptosis, monitored in parallel, was induced by myc-Bax and myc-Bcl-xAK, whereas myc- Bcl-xL diminished basal apoptotic rates, thus providing a proof on the function of the transfected proteins (data not shown). However, no direct interactions of the five representatives of the Bcl-2 family were seen with Bcl-xAK (Fig. 7A), thus suggesting that Bcl-xAK displays its activation of Bax and Bak in an indirect way via a not yet defined step. In this pathway Bcl-xAK and antiapoptotic family members act independent of each other on Bax and Bak (Fig. 7B). Discussion Pro- and antiapoptotic Bcl-2 proteins are critically involved in apoptosis regulation by controlling mitochondrial cell death PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34549