Regulation of Bcl-2 Proteins Independent of BH3 Figure 1. Efficient induction of cell death by Bcl-xAK. (A) The structure of the adenoviral construct AdV-AK is shown. The adenoviral E1 region was replaced by the Bcl-xAK cDNA driven by a tetracyclin-responsive promoter (PTRE), and the E3 region was replaced by the tetracyclin-controlled transactivator (tTA) driven by a CMV promoter (PCMV). The tTA mediates Tet-OFF regulation. Striped boxes indicate the poly(A)+ regions. (B) Bcl-xAK expression as determined by Western blot analysis is shown in melanoma cell lines SK-Mel-13, A-375 and Mel-2a at 48 h after transduction with AdV- AK (MOI = 50). Cells had received doxycycline (OFF condition) or were left without (ON condition). Equal protein loading was confirmed by b-actin. (C) Left, examples of cell cycle analysis after PI staining indicating sub-G1 apoptotic cell populations in Mel-2a at 48 h of transduction. Middle panel, detached and rounded cells indicating apoptosis are shown of Mel-2a at 48 h after transduction with AdV-AK under OFF and ON conditions. Right panel, chromatin condensation and nuclear fragmentation were visualized by bisbenzimide (DAPI) staining in Mel-2a at 48 h after AdV-AK transduction (MOI = 50). D–F) Time course analyses of apoptosis (D, flow cytometry after PI staining), cytotoxicity (E, LDH release) and cell proliferation (F, WST-1 assay) are shown for SK-Mel-13, A-375 and Mel-2a cells at 24, 48 and 72 h after transduction with AdV-AK (50 MOI, +Dox = Off, 2Dox = On). As positive controls for induced cytotoxicity, cell lines were completely lysed by triton X-100 (T = 100%) or were treated with doxorubicin (D, 500 nM, 72 h). WST-1 values are expressed as percent of non-treated controls ( = 100%). (G) For comparison, apoptosis induction (sub-G1 cells) by AdV-Nbk is shown for Mel-2a cells at 24 h, 48 h and 72 h (MOI = 50). AdV-Nbk shares the same backbone with AdV-AK. For induction, doxycycline was omitted (On). (H) A time course analysis of Bcl-xAK expression (3–48 h) after AdV-AK transduction and promoter induction is shown for Mel-2a, as determined by Western blot analysis. (I) Cell survival was determined according to calcein staining in Mel-2a cells at 48 h of Bcl-xAK induction. A shift to the left indicates calcein-negative ( = non viable) cells. (J) Quantification of the calcein experiment. (D, E, F, G, J) Means and standard deviations of triplicate values of representative experiments are shown. A luciferase-encoding adenovirus (Ad5-CMV-Luc) applied at the same MOI served as mock control (M), for controlling adenovirus transduction. All experiments were performed at least twice, resulting in highly comparable results. doi:10.1371/journal.pone.0034549.g001 (2.5 mM) or TMRM+ (1 mM), and changes of Dym were determined by flow cytometry. For measurement of intracellular ROS levels, the fluorescent dye H2DCFDA (29, 79- dichloro-dihydro-fluorescein-diacetate) was used. Cells were stained for 30 min with 15 mM H2DCFDA (Molecular Probes, Invitrogen), harvested by trypsinisation, resuspended in HBSS buffer (Biochrom, Berlin, Germany) and analyzed by flow cytometry. For ROS scavenging, N-acetyl cysteine (NAC, Sigma-Aldrich) was used in a concentration of 200 mM. Assays for Bax/Bak activation For determination of Bax and Bak clusters indicative for Bax/ Bak activation, DU145 cells were used, which had been stably transfected for expression of EGFP-Bax or EGFP-Bak, respectively [18]. Cells were seeded, transduced with AdV-AK (MOI = 50) and were cultured for 48 h with or without doxycycline. Bax and Bak clustering was demonstrated by a fluorescence microscope (Olympus BX50, Hamburg, Germany). For semi-quantitative evaluation, at least 500 cells of each condition were assessed. For analysis of Bax/Bak conformational changes related to activation, primary antibodies specific for Bax/Bak N-terminal domains were applied in flow cytometry (Bax-NT, Upstate, Lake Placid, USA, #06-499; Bak-NT, Merck, Darmstadt, Germany, #AM04). Melanoma cells (105) were harvested by trypsinisation and fixed for 30 min with 4% paraformaldehyde in PBS. Cells were suspended in saponin buffer (1% FCS, 0.1% saponin in PBS) and incubated for 1 h at 4uC in the dark with antibodies Bax-NT (1:100) or Bak-NT (1:10). As secondary antibodies, goat anti-rabbit IgG (H+L)-FITC (Jackson Immuno Research, West Grove, USA) and goat anti-mouse IgG (H+L)-FITC (SouthernBiotech, Birming- ham, AL, USA) were used. After washing and resuspension, cells were immediately measured by flow cytometry. Western blot analysis Detailed protocols for protein extraction and Western blot analysis had been described previously [22]. As a standard, 106 cells were harvested and dissolved in lysis buffer (150 mM NaCl, 1 mM EDTA, 0.5% SDS, 0.5% Nonidet P-40, 2 mM PMSF, 1 mM leupeptin, 1 mM pepstatin, 10 mM Tris-HCl, pH 7.5). For analysis of cytochrome c and mitochondrial localization of Bcl-2 proteins, cytosolic and mitochondrial cell fractions were separated by a mitochondria/cytosol fractionation kit (Alexis, Gr u¨nberg, Germany). The following primary antibodies were used: procaspase-3 (Cell Signaling, Danvers, MA, USA; rabbit; 1:1000), cleaved caspase-3 (Cell Signaling; rabbit; 1:1000), caspase-8 (Cell Signaling; mouse; 1:1000), caspase-9 (Cell Signaling; rabbit; 1:1000), Bcl-xL (Santa Cruz, Heidelberg, Germany; mouse; 1:200), mouse Bcl-2 (Santa Cruz; mouse; 1:200), human Bcl-2 (Santa Cruz; mouse; 1:200), Mcl-1 (Santa Cruz; rabbit; 1:200), Bax (Santa Cruz; rabbit; 1:200), Bak (Assay Biotechnology, Sunnyvale, CA, USA; rabbit; 1:500), Bad (Cell Signaling; rabbit; 1:1000), Puma (Epitomics, Burlin- game, CA USA; rabbit; 1:1000), Noxa (ProSci Incorporated, Poway, CA, USA; rabbit; 1:500), cytochrome c (BD Biosciences, Heidelberg, Germany; mouse; 1:1000), c-Myc (Calbiochem, Nottingham, UK; mouse; 1:500), anti-porin 31 HL (VDAC; Calbiochem; mouse; 1:5000), Glyceraldehyde 3-phosphate dehy- drogenase (GAPDH; Santa Cruz; mouse; 1:1000), b-actin (Sigma- Aldrich; mouse; 1:5000). As secondary antibodies, peroxidase- labeled goat anti-rabbit and goat anti-mouse antibodies were used (Dako, Hamburg, Germany; 1:5000). Immunoprecipitation with anti-Myc microbeads Melanoma cells (106, SK-Mel-13) were transiently transfected with plasmids encoding myc-tagged Bcl-2 proteins (0.1% DMRIE- C, 5 mg/ml plasmid). After 24 h (for Bcl-xL and Bax) or 48 h (for Bcl-xAK), cells were harvested, washed with ice-cold PBS and resuspended in 1 ml of pre-cooled lysis buffer (150 mM NaCl, 1% triton X-100, 50 mM Tris-HCl, pH 8). Microbeads covered with monoclonal anti-myc antibodies were given to the lysate for magnetic labelling of the tagged proteins. Beads and bound proteins were captured on flow-through magnetic columns, washed 46 with buffer 1 (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8) and washed for another time with 20 mM Tris-HCl (pH 7.5). Proteins were eluted with hot (95uC) elution buffer (50 mM DTT, 1% SDS, 1 mM EDTA, 0.005% bromphenol blue, 10% glycerol, 50 mM Tris-HCl, pH 6.8). No secondary antibodies were needed. The mock control were melanoma cells transiently transfected with an empty pcDNA3 plasmid. The mock control proved that the anti-Myc beads do not result in any non-specific precipitates. Immunoprecipitation of myc-tagged proteins was carried out with the mMACS c-myc-tagged protein isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Lysates and immunoprecipitates were investigated by Western blot analysis. Results Delayed but efficient apoptosis induction For investigating the efficacy and mechanism of Bcl-xAK- mediated apoptosis, an adenoviral vector was constructed with the PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34549