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RACK1 promotes Bax oligomerization and dissociates the interaction of Bax and Bcl-XL
Cellular Signalling 22 (2010) 1495–1501
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
RACK1 promotes Bax oligomerization and dissociates the interaction of Bax and Bcl-XL Yinyuan Wu a, Yinyin Wang a, Yang Sun a, Liying Zhang b, Dianjun Wang c, Fangli Ren a, Donald Chang b, Zhijie Chang a,⁎, Baoqing Jia c,⁎ a School of Medicine, Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing (100084), China b Department of Biology, Hong Kong University of Science & Technology, Hong Kong, China c Department of General Surgery, Chinese PLA General Hospital, Beijing (100853), China
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Article history: Received 2 March 2010 Received in revised form 25 May 2010 Accepted 29 May 2010 Available online 10 June 2010 Keywords: RACK1 Bax oligomerization Bcl-XL Apoptosis
a b s t r a c t Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK1 inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Apoptosis is a regulated mechanism to selectively eliminate unwanted or damaged cells. Apoptosis is induced by mitochondriadependent and -independent pathways. In mitochondria-dependent induced apoptosis, Bcl-2 family members are major regulators, which function as proapoptotic or anti-apoptotic factors. The proapoptotic members consist of the proteins with Bcl-2 homology (BH) 1, 2 and 3 domains, including Bax, Bak, and Bok, and the proteins with only the BH3 domain, including Bad, Bid, Bik, Bim, and Hrk [1–3]. The antiapoptotic members include Bcl-2, Bcl-XL, Ced-9, Bcl-w, and Mcl-1, which display sequence conservation throughout all four BH domains (BH1-4) [2]. It has been demonstrated that Bax plays an essential role in apoptosis, as revealed by gene disruption experiments [4–7]. The mechanism for the functions of Bax in apopotosis has been well studied. Bax is localized mostly in the cytoplasm, but redistributes to the mitochondria in response to cell death stimuli [8–10]. After translocation to mitochondria, Bax induces cytochrome c release ⁎ Corresponding authors. Chang is to be contacted at Tel.: +86 10 62785076; fax: +86 10 62773624. Jia, Tel.: +86 10 66938126. E-mail addresses: [email protected] (Z. Chang), [email protected] (B. Jia). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.05.018
either by forming a pore after oligomerization in the outer mitochondrial membrane, or by opening other channels [11–13]. Apoptosis is a very important decision for cells and has to be regulated precisely. As Bax is a critical factor to induce the release of cytochrome c to initiate apoptosis, the most important regulation for apoptosis is on the Bax activity. Several pathways have been revealed to regulate the Bax activity. For example, JNK, through phosphorylation of 14-3-3 proteins, promotes Bax translocation to the mitochondria, resulting in an accelerated apoptosis [14]. p53 directly interacts with Bax to promote Bax oligomerization [15]. The proapoptotic protein Bid and Bim act in a “hit-and-run” manner to induce Bax conformation changed, triggering Bax oligomerization and integration into the mitochondrial membranes [16–18]. On the other hand, the anti-apoptotic factor Bcl-XL, which interacts with Bax, can sequester Bax to prevent its oligomerization. The mechanisms underlying Bax oligomerization, however, are not fully understood. RACK1 was originally identified as an anchoring protein for βIIPKC to increase PKC kinase activity [19]. RACK1 is a seven WD motifcontaining protein with numerous downstream effectors regulating various cellular functions [20]. To date, RACK1 has been found to play different roles in apoptosis. Several groups reported that RACK1 can suppress apoptosis to regulate cell growth [21–23]. Controversially, RACK1 was reported to induce apoptosis of colon cancer cells through
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suppression of Src activity [24]. The detailed roles of RACK1 on the regulation of apoptosis remain to be revealed. In this study we identified that RACK1 interacts with Bax by a yeast two-hybrid assay. We observed that overexpression of RACK1 promotes UV-induced apoptosis. We provide evidence that RACK1 enhances apoptosis by promoting Bax oligomerization and dissociating the Bax/Bcl-XL complex. 2. Results 2.1. Bax interacts with RACK1 through BH3 domain both in vivo and in vitro To identify possible additional partner protein(s) interacting with Bax, we performed a yeast two-hybrid screen using Bax as bait. We identified 20 positive clones and confirmed the interaction by a liquid β-Gal assay (data not shown). After sequencing all of the 20 positive clones, we identified two positive clones coding for the RACK1 protein, which was previously characterized as one of a group of PKCinteracting proteins [18]. To confirm the interaction of Bax and RACK1 in mammalian cells, we performed an immunoprecipitation experiment by co-expressing Myc-RACK1 and Flag-Bax in HEK293T cells, and immunoprecipitating the cell lysates with an anti-Flag antibody or an anti-Myc antibody. A Western blotting of the precipitates indicated that MycRACK1 is co-immunoprecipitated with Flag-Bax and Flag-Bax is coimmunoprecipitated with Myc-RACK1 (Fig. 1A). To explore a possible interaction between RACK1 and Bax in vitro, we purified the GST-RACK1 protein and performed a GST pull down experiment. The results showed that the Flag-Bax protein is pulled down with GST-RACK1, but not GST (Fig.1B, top panel, lane 2). These data indicate that Bax interacts with RACK1 both in vivo and in vitro. We next delineated the regions of Bax that interacts with RACK1 using a series of truncated Bax proteins tagged with YFP and the full length Bax tagged with GFP. Myc-RACK1 was co-expressed with all the truncated proteins in HEK 293 T cells and coimmunoprecipitated with an anti-GFP antibody, which recognizes both YFP and GFP. The results demonstrated that the BH3 domain of Bax is responsible for the interaction of Bax and RACK1 (Fig.1C, top panel, lanes 2, 3, 7). 2.2. RACK1 colocalizes with Bax and promotes Bax oligomerization directly Bax has been reported to form oligomers during apoptosis [12,25–27]. To investigate the functional consequence of the interaction of Bax with RACK1, we examined whether RACK1 is involved in the formation of Bax oligomerization. To this end, we first examined whether RACK1 could colocalize with Bax in the intact cells. We performed an immunostaining experiment by co-expressing GFP-Bax and Myc-RACK1 in HeLa cells. The results showed that when Bax is expressed alone, it distributes almost in the whole cells (Fig. 2A), but when Bax and RACK1 are co-expressed, Bax integrates into a punctual structure and colocalizes with RACK1 (Fig. 2B). The cell numbers with integrated Bax when Bax and RACK1 are co-expressed are much more than that when only Bax is overexpressed (Fig. 2C). The integrated structure of Bax under RACK1 co-expression is reminiscent of Bax oligomerization [17]. To further determine whether RACK1 is involved in Bax oligomerization, we performed an immunoprecipitation experiment by coexpressing Myc-RACK1, Flag-Bax and Myc-Bax in HEK293T cells. A Western blotting of the precipitates with an anti-Myc antibody showed that Myc-Bax was co-immunoprecipitated with Flag-Bax in the presence of Myc-RACK1, indicating Bax forms oligomers by RACK1 (Fig. 2D, top right panel, lane 2). At the same time, we observed that Myc-RACK1 was precipitated by Flag-Bax (Fig. 2D, bottom right panel, lane 2), which is consistent with the results in Fig. 1A. We also
Fig. 1. Interaction of RACK1 with Bax in vivo and in vitro. (A) RACK1 interacts with Bax in vivo. Cells were co-transfected with Myc-RACK1 and Flag-Bax. Immunoprecipitation was performed using an anti-M2 or anti-Myc antibody and the precipitants were detected using an anti-Myc or anti-M2 antibody. (B) RACK1 interacts with Bax in vitro. A GST pull down assay was performed with the purified GST or GST-RACK1 protein and Flag-Bax protein expressed in HEK293T cells. (C) The BH3 domain interacts with RACK1. Schematic illustration of Bax and the domains is shown. The full length and six domains of Bax were co-expressed with Myc-RACK1. Immunprecipitation was performed by using an anti-GFP antibody. The precipitants were detected with an anti-Myc antibody.
performed an in vitro experiment by adding GST or GST-RACK1 to the cell lysates containing Flag-Bax and Myc-Bax. The result showed that Flag-Bax is co-immunoprecipitated with Myc-Bax in the presence of GST-RACK1 (Fig. 2E, top right panel, lane 2), but not in the presence of GST (Fig. 2E, top right panel, lane 1). Reciprocally, MycBax is co-immunoprecipitated with Flag-Bax in the presence of GSTRACK1 (Fig. 2E, bottom right panel, lane 2). These results together with the immunostaining data indicate that RACK1 promotes Bax oligomerization.
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Fig. 2. RACK1 colocalizes with Bax and promotes Bax oligomerization. (A) The location of Bax and RACK1 in HeLa cells. HeLa cells were transfected with GFP-Bax or Myc-RACK1, and stained with an anti-Myc (red) antibody. (B) RACK1 colocalizes with Bax. HeLa cells co-expressing Myc-RACK1 and GFP-Bax were stained with an anti-Myc (red) antibody. The images were viewed with a confocal microscope. The co-localization of Myc-RACK1 and GFP-Bax is showed as yellow color. Scale bar, 10 μm. (C) A quantitative illustration of cells with co-localized Bax and RACK1. HeLa cells transfected with the indicated plasmids and assessed by counting the number of cells exhibiting Bax and RACK1 co-localization. (D) RACK1 promotes Bax oligomerization in vivo. The cells were transfected with Flag-Bax and Myc-Bax with or without Myc-RACK1. The Bax oligomerizaton is revealed by an immunoprecipitation of Flag-Bax and Myc-Bax, which was performed using an anti-M2 antibody and the precipitants were detected using an anti-Myc antibody. (E) RACK1 promotes Bax oligomerization in vitro. Purified GST or GST-RACK1 protein was added to the cell lysates with Flag-Bax protein and Myc-Bax expressed in HEK293T cells. Immunoprecipitation was performed using an anti-M2 (or reciprocally anti-Myc) antibody and the precipitants were detected using an anti-Myc (or anti-M2) antibody.
2.3. RACK1 dissociates the complex of Bax and Bcl-XL Bcl-XL is known to sequester Bax by forming a complex and to inhibit Bax oligomerization during apoptosis [9]. We speculated that
RACK1 might interact with Bcl-XL. To test this hypothesis, we performed an immunoprecipitation experiment by co-expressing Myc-RACK1 and GFP-Bcl-XL in HEK293T cells. The results indicated that Myc-RACK1 was co-immunoprecipitated with GFP-Bcl-XL
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(Fig. 3A, top right panel, lane 3) and vice versa (Fig. 3A, bottom right panel, lane 3). To further confirm the possible interaction between RACK1 and Bcl-XL in vitro, we performed a GST pull down experiment. The results showed that the GFP-Bcl-XL protein was pulled down with GST-RACK1, but not with GST alone (Fig. 3B, top panel, lane 2). These data indicate that RACK1 interacts with Bcl-XL both in vivo and in vitro. Because RACK1 interacts with both Bax and Bcl-XL, we speculated that RACK1 might cause dissociation of Bax and Bcl-XL through binding to Bcl-XL. A reciprocal immunoprecipitation result indicated that the association of Bax and Bcl-XL is decreased in the RACK1 overexpressed cells (Fig. 3C, comparing right lanes 2 to 1), while it is enhanced in the RACK1 depleted cells (Fig. 3C, comparing right lanes 4 to 3). We also observed that both Flag-Bax and GFP-Bcl-XL precipitated down Myc-RACK1. These data suggest that RACK1 disrupts the interaction of Bax and Bcl-XL. To further confirm the effect of RACK1 on the interaction of Bax and Bcl-XL in vitro, we performed an immunoprecipitation experiment in the presence of
GST or GST-RACK1. The result showed that less amount of Flag-Bax is co-immunoprecipitated with GFP-Bcl-XL in the presence of GSTRACK1 than that in the presence of GST (Fig. 3D, top right panel). Similar results were obtained in the same cell lysates co-precipitated by M2 antibody (Fig. 3D, bottom right panel). All the data indicate that RACK1 interrupts the interaction of Bax and Bcl-XL. 2.4. RACK1 promotes UV-induced apoptosis The above data demonstrated that RACK1 promotes Bax oligomerization, which is a critical process in apoptosis. To address whether RACK1 is involved in apoptosis, we used an annexin staining and flow cytometry method as a sensitive, quantitative assay to examine cell apoptosis in both early and late apoptosis [28]. We observed that more RACK1-overexpressing cells were undergoing apoptosis (25.8%) than the mock cells (4.1%) (Fig. 4A). Consistent with these results, depleting RACK1 also reduces UV-induced apoptosis while overexpressing RACK1 increases the effect (Fig. 4B and C).
Fig. 3. RACK1 dissociates Bax from Bcl-XL by binding to Bcl-XL. (A) RACK1 interacts with Bcl-XL in vivo. The HEK293T cells expressing Myc-RACK1 and GFP-Bcl-XL were immunoprecipitated with an anti-GFP (or anti-Myc) antibody, and the precipitants were detected using an anti-Myc (or anti-GFP) antibody. (B) RACK1 interacts with Bcl-XL in vitro. A GST pull-down assay was performed with the purified GST or GST-RACK1 protein and GFP-Bcl-XL protein expressed in HEK293T cells. The precipitated GFP-Bcl-XL protein was examined by a GFP antibody. (C) RACK1 disrupts the association of Bax and Bcl-XL in vivo. The complex of Bax and Bcl-XL was examined by an immunoprecipitation experiment using GFP antibody (or anti-M2) in the HEK293T cells with overexpressed (Myc-RACK1) and depleted (siRNA) RACK1. (D) RACK1 disrupts the association of Bax and Bcl-XL in vitro. A reciprocal immunoprecipitation experiment revealing the interaction of Bax and Bcl-XL was performed with expression of Flag-Bax and GFP-Bcl-XL in HEK293T cells in the presence of the purified GST or GST-RACK1 protein by using an anti-GFP or M2 antibody.
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Fig. 4. RACK1 promotes apoptosis. (A) Effect of RACK1 overexpression on apoptosis, as measured by annexin V/PI staining and flow cytometry. Hela cells were stained with annexin V–FITC and PI and analyzed by flow cytometry after transfection with the empty vector, or Myc-RACK1 for 24 h. Cells undergoing early apoptosis (annexin positive and PI negative) are located in the right lower box. Cells in the late stage of apoptosis and dead cells (annexin and PI positive) are located in the right upper box. (B) Overexpression of RACK1 enhanced UV-induced apoptosis. Hela cells were transfected with the empty vector, or Myc-RACK1 for 24 h. The cells were treated with UV irradiation at a fluence of 120 mJ/cm2. 5 h after UV irradiation, cells were stained with annexin V–FITC and PI and analyzed by flow cytometry. (C) Depletion of RACK1 reduced UV-induced apoptosis. Hela cells were transfected with the control siRNA, or siRNA targeting RACK1. The cells were treated the same way as in (B). Data are representative of three independent experiments.
Collectively, these results suggest that RACK1 promotes apoptosis in the Hela cells. 3. Discussion The Bcl-2 family proteins play a critical role in the apoptotic process. Among the Bcl-2 family proteins, Bax is thought to be the most important proapoptotic protein [6,7]. In normal conditions, Bax
is mainly localized in the cytosol. Upon apoptotic stimuli, Bax undergoes a conformational change and translocates to the outer mitochondrial membrane [8]. Then, Bax becomes oligomerized and permeabilizes the mitochondrial membrane to release apoptotic factors [29,30]. Currently, there is a great interest in understanding how the Bax activity is regulated during apoptosis. It has been shown that several proteins, including Mcl-1, Ku70, 14-3-3 and Humanin, sequester Bax in the cytoplasm [31–35]. Several anti-apoptotic Bcl-2
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family proteins, such as Bcl-2, Bcl-XL in the mitochondria, also inhibit Bax translocation and activation [36,37]. However, under certain apoptotic stimuli, Bax can be activated directly by certain BH3-only proteins (e.g. tBid and Bim) and undergoes a conformational change [14,16,17,38,39]. In this study, we use a yeast two-hybrid assay to identify RACK1 as a novel binding partner of Bax. We confirmed that RACK1 interacts with Bax in mammalian cells and found that RACK1 promotes Bax oligomerization. Our study provided a new mechanism for the regulations of Bax oligomerization. The balance between pro- and anti-apoptotic proteins helps determine the susceptibility of cells in response to a cell death signal. Bax with Bcl-XL are two key pro- and anti-apoptotic proteins. Bcl-XL is known to interact with Bax and inhibit Bax oligomerization during apoptosis [9]. In this study, we observed that the interaction of Bax with Bcl-XL is interrupted by overexpressed RACK1, and the interaction of Bax with Bcl-XL is enhanced in cells in depletion of RACK1 (Fig.3C and D). These results indicated that RACK1 inhibits the interaction of Bax and Bcl-XL. In an apoptosis experiment, we observed that RACK1-overexpressing cells undergo apoptosis while depleting RACK1 reduces UV-induced apoptosis. Therefore, we proposed that RACK1 functions in Bax oligomerization to induce apoptosis. RACK1 has been reported as an E3 ligase component to mediate the degradation of DeltaNp63alpha, a member of the p53 family [40] and to interact with Elongin C and mediate the degradation of HIF-1α [41]. In our experiments, we did not observe any effect of protein degradation of Bax and Bcl-XL under RACK1 overexpression (data not shown). We explain that RACK1 is not an E3 ligase although it can participate in the formation of an E3 ligase complex. When it functions in forming an E3 ligase complex, RACK1 may require an E3 ligase. However, in the situation of Bax, RACK1 cannot recruit any E3 ligase. Therefore RACK1 could not mediate the degradation of Bax. Our result that RACK1 promotes Bax oligomerization to induce apoptosis in Hela cells is consistent with the observations in the HT-29 cells [24]. Especially, our observation that RACK1 promotes the oligomerizatioin of Bax examined by immunostaining experiment is almost the same as the observation from a previous report (comparing the Fig. 3B in literature [24] with our results in Fig. 2B). In this study, we have provided further evidence that RACK1 interacts with Bax and promotes the oligomerization of Bax by immunoprecipitation experiment. Manidipudi V and Cartwright CA analyzed that RACK1 inhibits the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL and induces the expression of proapoptotic protein Bim [24]. Our study provides another mechanism that RACK1 promotes apoptosis by disassociating the interaction of Bax with Bcl-XL, which is similar to the mechanism by which p53 regulates the activity of Bax [15]. However, other groups reported that RACK1 inhibits apoptosis [21–23]. These controversial observations demonstrated a diverse role of RACK1 in different tumors with different target proteins. Indeed, RACK1 contains 7 WD domains which mediate the association of different substrates. We believe that the roles of RACK1 on cell survival or death are dependent on the dominance of different targeted proteins. Further study is required to reveal the detailed functions of RACK1 in different cells. 4. Materials and methods 4.1. Plasmids and reagents pcDNA3.1/Myc-RACK1 and pGEX4T-2/GST-RACK1 were generated by inserting the PCR-amplified related inserts into pcDNA3.1/Myc and pGEX4T-2. pcDNA3.1/Myc-Bax and pcDNA3.1/Flag-Bax were generated by inserting the PCR-amplified related inserts into a pcDNA3.1/ Myc and pcDNA3.1/Flag. Constructs expressing GFP-Bax, YFP-ART, YFP-ART-BH3, YFP-BH3, YFP-α5/6, YFP-α5/6-TM, YFP-TM, and GFPBcl-XL were kindly provided by Dr. Donald Chang, Hong Kong University of Science & Technology, Hong Kong, China. pcDNA3.1/
Myc, pcDNA3.1/Flag and pGEX4T-2 were kept in this lab. Antibody against Flag (M2) was from Sigma. Antibodies against Myc (9E10) and GFP were purchased from Santa Cruz. Fluorescent secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. The target sequences of siRNA are from 146 to 166 bp (human RACK1 sequence accession no. NM_006098), which is confirmed by Cartwright [42]. 4.2. Cell culture HEK293T and Hela cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. All the cells were kept at 37 °C in 5% CO2-containing atmosphere. The medium and serum were purchased from Gibco. For UV treatment, medium was removed and saved, cells were rinsed with PBS and irradiated, and medium was restored. Cells were exposed to UV irradiation at a fluence of 120 mJ/cm2 and observed at 5 h after treatment. 4.3. Co-immunoprecipitations HEK293T cells were plated in 60 mm dishes the day before transfection. 5 μg of pcDNA3.1/Myc-RACK1 and the indicated plasmids were transfected. If necessary, pcDNA3.1/Myc or pcDNA3.1/Flag empty vector was added to ensure that an equal amount of plasmid was transfected each dish. After transfection for 18-36 h, cells were lysed in 600 μl cell lysis buffer (50 mM Tris·Cl, 150 mM NaCl, 50 mM NaF, 0.5% NP-40, pH 7.5) with protease inhibitors to prepare wholecell lysates. The lysate was mixed with proper antibodies and incubated at 4 °C for 2 h, followed by addition of protein G-agarose beads to pellet the immune complex. The immunoprecipitants and 5% of lysates were analyzed by immunoblotting for the indicated proteins. 4.4. GST pull down assays GST-fused RACK1 (GST-RACK1) was expressed in E. coli strain BL21 cells and purified by affinity chromatography with glutathioneSepharose 4B resin (GE Healthcare). 50 μg of each purified protein was mixed with glutathione-Sepharose beads and incubated at 4 °C for 30 min before use. Constructs expressing the indicated plasmids were tansfected into HEK293T cells individually. After transfection for 18–36 h, the whole-cell lysates were prepared and incubated with appropriate GST fusion protein/glutathione-Sepharose beads complex at 4 °C for 2 h. The precipitates were analyzed by immunoblotting for the indicated proteins. 4.5. Immunofluorescent staining and microscopy Hela cells were cultured on 6-well plates with 8 × 104 cells per well (Corning Incorporated, Corning, NY). Cells were transfected with the indicated plasmids and fixed after 36 h transfection with 4% paraformaldehyde for 20 min and perforated with 0.3% Triton X-100 for 10 min. The cells were blocked with 10% FBS for 50 min followed by incubation with c-Myc antibody at 4 °C overnight. The cells were incubated with a secondary antibody conjugated with goat antimouse IgG/TRITC antibody (Jackson Research Laboratories) for 1 h and counterstained with DAPI for 10 min. Finally, the results were visualized by a confocal laser scanning microscope (OLYMPUS BX61). The co-localization of the two proteins was shown as a merged image. 4.6. Flow cytometry To quantify cells in early and late apoptosis, transfectants were incubated with annexin V–FITC/PI for 10–15 min at RT, according to the manufacturer's protocol (Pharmingen) and analyzed by flow cytometry. Cells (104) were analyzed by an FACScan cell sorter (Becton-Dickinson,
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
RACK1 promotes Bax oligomerization and dissociates the interaction of Bax and Bcl-XL Yinyuan Wu a, Yinyin Wang a, Yang Sun a, Liying Zhang b, Dianjun Wang c, Fangli Ren a, Donald Chang b, Zhijie Chang a,⁎, Baoqing Jia c,⁎ a School of Medicine, Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing (100084), China b Department of Biology, Hong Kong University of Science & Technology, Hong Kong, China c Department of General Surgery, Chinese PLA General Hospital, Beijing (100853), China
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Article history: Received 2 March 2010 Received in revised form 25 May 2010 Accepted 29 May 2010 Available online 10 June 2010 Keywords: RACK1 Bax oligomerization Bcl-XL Apoptosis
a b s t r a c t Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK1 inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Apoptosis is a regulated mechanism to selectively eliminate unwanted or damaged cells. Apoptosis is induced by mitochondriadependent and -independent pathways. In mitochondria-dependent induced apoptosis, Bcl-2 family members are major regulators, which function as proapoptotic or anti-apoptotic factors. The proapoptotic members consist of the proteins with Bcl-2 homology (BH) 1, 2 and 3 domains, including Bax, Bak, and Bok, and the proteins with only the BH3 domain, including Bad, Bid, Bik, Bim, and Hrk [1–3]. The antiapoptotic members include Bcl-2, Bcl-XL, Ced-9, Bcl-w, and Mcl-1, which display sequence conservation throughout all four BH domains (BH1-4) [2]. It has been demonstrated that Bax plays an essential role in apoptosis, as revealed by gene disruption experiments [4–7]. The mechanism for the functions of Bax in apopotosis has been well studied. Bax is localized mostly in the cytoplasm, but redistributes to the mitochondria in response to cell death stimuli [8–10]. After translocation to mitochondria, Bax induces cytochrome c release ⁎ Corresponding authors. Chang is to be contacted at Tel.: +86 10 62785076; fax: +86 10 62773624. Jia, Tel.: +86 10 66938126. E-mail addresses: [email protected] (Z. Chang), [email protected] (B. Jia). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.05.018
either by forming a pore after oligomerization in the outer mitochondrial membrane, or by opening other channels [11–13]. Apoptosis is a very important decision for cells and has to be regulated precisely. As Bax is a critical factor to induce the release of cytochrome c to initiate apoptosis, the most important regulation for apoptosis is on the Bax activity. Several pathways have been revealed to regulate the Bax activity. For example, JNK, through phosphorylation of 14-3-3 proteins, promotes Bax translocation to the mitochondria, resulting in an accelerated apoptosis [14]. p53 directly interacts with Bax to promote Bax oligomerization [15]. The proapoptotic protein Bid and Bim act in a “hit-and-run” manner to induce Bax conformation changed, triggering Bax oligomerization and integration into the mitochondrial membranes [16–18]. On the other hand, the anti-apoptotic factor Bcl-XL, which interacts with Bax, can sequester Bax to prevent its oligomerization. The mechanisms underlying Bax oligomerization, however, are not fully understood. RACK1 was originally identified as an anchoring protein for βIIPKC to increase PKC kinase activity [19]. RACK1 is a seven WD motifcontaining protein with numerous downstream effectors regulating various cellular functions [20]. To date, RACK1 has been found to play different roles in apoptosis. Several groups reported that RACK1 can suppress apoptosis to regulate cell growth [21–23]. Controversially, RACK1 was reported to induce apoptosis of colon cancer cells through
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suppression of Src activity [24]. The detailed roles of RACK1 on the regulation of apoptosis remain to be revealed. In this study we identified that RACK1 interacts with Bax by a yeast two-hybrid assay. We observed that overexpression of RACK1 promotes UV-induced apoptosis. We provide evidence that RACK1 enhances apoptosis by promoting Bax oligomerization and dissociating the Bax/Bcl-XL complex. 2. Results 2.1. Bax interacts with RACK1 through BH3 domain both in vivo and in vitro To identify possible additional partner protein(s) interacting with Bax, we performed a yeast two-hybrid screen using Bax as bait. We identified 20 positive clones and confirmed the interaction by a liquid β-Gal assay (data not shown). After sequencing all of the 20 positive clones, we identified two positive clones coding for the RACK1 protein, which was previously characterized as one of a group of PKCinteracting proteins [18]. To confirm the interaction of Bax and RACK1 in mammalian cells, we performed an immunoprecipitation experiment by co-expressing Myc-RACK1 and Flag-Bax in HEK293T cells, and immunoprecipitating the cell lysates with an anti-Flag antibody or an anti-Myc antibody. A Western blotting of the precipitates indicated that MycRACK1 is co-immunoprecipitated with Flag-Bax and Flag-Bax is coimmunoprecipitated with Myc-RACK1 (Fig. 1A). To explore a possible interaction between RACK1 and Bax in vitro, we purified the GST-RACK1 protein and performed a GST pull down experiment. The results showed that the Flag-Bax protein is pulled down with GST-RACK1, but not GST (Fig.1B, top panel, lane 2). These data indicate that Bax interacts with RACK1 both in vivo and in vitro. We next delineated the regions of Bax that interacts with RACK1 using a series of truncated Bax proteins tagged with YFP and the full length Bax tagged with GFP. Myc-RACK1 was co-expressed with all the truncated proteins in HEK 293 T cells and coimmunoprecipitated with an anti-GFP antibody, which recognizes both YFP and GFP. The results demonstrated that the BH3 domain of Bax is responsible for the interaction of Bax and RACK1 (Fig.1C, top panel, lanes 2, 3, 7). 2.2. RACK1 colocalizes with Bax and promotes Bax oligomerization directly Bax has been reported to form oligomers during apoptosis [12,25–27]. To investigate the functional consequence of the interaction of Bax with RACK1, we examined whether RACK1 is involved in the formation of Bax oligomerization. To this end, we first examined whether RACK1 could colocalize with Bax in the intact cells. We performed an immunostaining experiment by co-expressing GFP-Bax and Myc-RACK1 in HeLa cells. The results showed that when Bax is expressed alone, it distributes almost in the whole cells (Fig. 2A), but when Bax and RACK1 are co-expressed, Bax integrates into a punctual structure and colocalizes with RACK1 (Fig. 2B). The cell numbers with integrated Bax when Bax and RACK1 are co-expressed are much more than that when only Bax is overexpressed (Fig. 2C). The integrated structure of Bax under RACK1 co-expression is reminiscent of Bax oligomerization [17]. To further determine whether RACK1 is involved in Bax oligomerization, we performed an immunoprecipitation experiment by coexpressing Myc-RACK1, Flag-Bax and Myc-Bax in HEK293T cells. A Western blotting of the precipitates with an anti-Myc antibody showed that Myc-Bax was co-immunoprecipitated with Flag-Bax in the presence of Myc-RACK1, indicating Bax forms oligomers by RACK1 (Fig. 2D, top right panel, lane 2). At the same time, we observed that Myc-RACK1 was precipitated by Flag-Bax (Fig. 2D, bottom right panel, lane 2), which is consistent with the results in Fig. 1A. We also
Fig. 1. Interaction of RACK1 with Bax in vivo and in vitro. (A) RACK1 interacts with Bax in vivo. Cells were co-transfected with Myc-RACK1 and Flag-Bax. Immunoprecipitation was performed using an anti-M2 or anti-Myc antibody and the precipitants were detected using an anti-Myc or anti-M2 antibody. (B) RACK1 interacts with Bax in vitro. A GST pull down assay was performed with the purified GST or GST-RACK1 protein and Flag-Bax protein expressed in HEK293T cells. (C) The BH3 domain interacts with RACK1. Schematic illustration of Bax and the domains is shown. The full length and six domains of Bax were co-expressed with Myc-RACK1. Immunprecipitation was performed by using an anti-GFP antibody. The precipitants were detected with an anti-Myc antibody.
performed an in vitro experiment by adding GST or GST-RACK1 to the cell lysates containing Flag-Bax and Myc-Bax. The result showed that Flag-Bax is co-immunoprecipitated with Myc-Bax in the presence of GST-RACK1 (Fig. 2E, top right panel, lane 2), but not in the presence of GST (Fig. 2E, top right panel, lane 1). Reciprocally, MycBax is co-immunoprecipitated with Flag-Bax in the presence of GSTRACK1 (Fig. 2E, bottom right panel, lane 2). These results together with the immunostaining data indicate that RACK1 promotes Bax oligomerization.
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Fig. 2. RACK1 colocalizes with Bax and promotes Bax oligomerization. (A) The location of Bax and RACK1 in HeLa cells. HeLa cells were transfected with GFP-Bax or Myc-RACK1, and stained with an anti-Myc (red) antibody. (B) RACK1 colocalizes with Bax. HeLa cells co-expressing Myc-RACK1 and GFP-Bax were stained with an anti-Myc (red) antibody. The images were viewed with a confocal microscope. The co-localization of Myc-RACK1 and GFP-Bax is showed as yellow color. Scale bar, 10 μm. (C) A quantitative illustration of cells with co-localized Bax and RACK1. HeLa cells transfected with the indicated plasmids and assessed by counting the number of cells exhibiting Bax and RACK1 co-localization. (D) RACK1 promotes Bax oligomerization in vivo. The cells were transfected with Flag-Bax and Myc-Bax with or without Myc-RACK1. The Bax oligomerizaton is revealed by an immunoprecipitation of Flag-Bax and Myc-Bax, which was performed using an anti-M2 antibody and the precipitants were detected using an anti-Myc antibody. (E) RACK1 promotes Bax oligomerization in vitro. Purified GST or GST-RACK1 protein was added to the cell lysates with Flag-Bax protein and Myc-Bax expressed in HEK293T cells. Immunoprecipitation was performed using an anti-M2 (or reciprocally anti-Myc) antibody and the precipitants were detected using an anti-Myc (or anti-M2) antibody.
2.3. RACK1 dissociates the complex of Bax and Bcl-XL Bcl-XL is known to sequester Bax by forming a complex and to inhibit Bax oligomerization during apoptosis [9]. We speculated that
RACK1 might interact with Bcl-XL. To test this hypothesis, we performed an immunoprecipitation experiment by co-expressing Myc-RACK1 and GFP-Bcl-XL in HEK293T cells. The results indicated that Myc-RACK1 was co-immunoprecipitated with GFP-Bcl-XL
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(Fig. 3A, top right panel, lane 3) and vice versa (Fig. 3A, bottom right panel, lane 3). To further confirm the possible interaction between RACK1 and Bcl-XL in vitro, we performed a GST pull down experiment. The results showed that the GFP-Bcl-XL protein was pulled down with GST-RACK1, but not with GST alone (Fig. 3B, top panel, lane 2). These data indicate that RACK1 interacts with Bcl-XL both in vivo and in vitro. Because RACK1 interacts with both Bax and Bcl-XL, we speculated that RACK1 might cause dissociation of Bax and Bcl-XL through binding to Bcl-XL. A reciprocal immunoprecipitation result indicated that the association of Bax and Bcl-XL is decreased in the RACK1 overexpressed cells (Fig. 3C, comparing right lanes 2 to 1), while it is enhanced in the RACK1 depleted cells (Fig. 3C, comparing right lanes 4 to 3). We also observed that both Flag-Bax and GFP-Bcl-XL precipitated down Myc-RACK1. These data suggest that RACK1 disrupts the interaction of Bax and Bcl-XL. To further confirm the effect of RACK1 on the interaction of Bax and Bcl-XL in vitro, we performed an immunoprecipitation experiment in the presence of
GST or GST-RACK1. The result showed that less amount of Flag-Bax is co-immunoprecipitated with GFP-Bcl-XL in the presence of GSTRACK1 than that in the presence of GST (Fig. 3D, top right panel). Similar results were obtained in the same cell lysates co-precipitated by M2 antibody (Fig. 3D, bottom right panel). All the data indicate that RACK1 interrupts the interaction of Bax and Bcl-XL. 2.4. RACK1 promotes UV-induced apoptosis The above data demonstrated that RACK1 promotes Bax oligomerization, which is a critical process in apoptosis. To address whether RACK1 is involved in apoptosis, we used an annexin staining and flow cytometry method as a sensitive, quantitative assay to examine cell apoptosis in both early and late apoptosis [28]. We observed that more RACK1-overexpressing cells were undergoing apoptosis (25.8%) than the mock cells (4.1%) (Fig. 4A). Consistent with these results, depleting RACK1 also reduces UV-induced apoptosis while overexpressing RACK1 increases the effect (Fig. 4B and C).
Fig. 3. RACK1 dissociates Bax from Bcl-XL by binding to Bcl-XL. (A) RACK1 interacts with Bcl-XL in vivo. The HEK293T cells expressing Myc-RACK1 and GFP-Bcl-XL were immunoprecipitated with an anti-GFP (or anti-Myc) antibody, and the precipitants were detected using an anti-Myc (or anti-GFP) antibody. (B) RACK1 interacts with Bcl-XL in vitro. A GST pull-down assay was performed with the purified GST or GST-RACK1 protein and GFP-Bcl-XL protein expressed in HEK293T cells. The precipitated GFP-Bcl-XL protein was examined by a GFP antibody. (C) RACK1 disrupts the association of Bax and Bcl-XL in vivo. The complex of Bax and Bcl-XL was examined by an immunoprecipitation experiment using GFP antibody (or anti-M2) in the HEK293T cells with overexpressed (Myc-RACK1) and depleted (siRNA) RACK1. (D) RACK1 disrupts the association of Bax and Bcl-XL in vitro. A reciprocal immunoprecipitation experiment revealing the interaction of Bax and Bcl-XL was performed with expression of Flag-Bax and GFP-Bcl-XL in HEK293T cells in the presence of the purified GST or GST-RACK1 protein by using an anti-GFP or M2 antibody.
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Fig. 4. RACK1 promotes apoptosis. (A) Effect of RACK1 overexpression on apoptosis, as measured by annexin V/PI staining and flow cytometry. Hela cells were stained with annexin V–FITC and PI and analyzed by flow cytometry after transfection with the empty vector, or Myc-RACK1 for 24 h. Cells undergoing early apoptosis (annexin positive and PI negative) are located in the right lower box. Cells in the late stage of apoptosis and dead cells (annexin and PI positive) are located in the right upper box. (B) Overexpression of RACK1 enhanced UV-induced apoptosis. Hela cells were transfected with the empty vector, or Myc-RACK1 for 24 h. The cells were treated with UV irradiation at a fluence of 120 mJ/cm2. 5 h after UV irradiation, cells were stained with annexin V–FITC and PI and analyzed by flow cytometry. (C) Depletion of RACK1 reduced UV-induced apoptosis. Hela cells were transfected with the control siRNA, or siRNA targeting RACK1. The cells were treated the same way as in (B). Data are representative of three independent experiments.
Collectively, these results suggest that RACK1 promotes apoptosis in the Hela cells. 3. Discussion The Bcl-2 family proteins play a critical role in the apoptotic process. Among the Bcl-2 family proteins, Bax is thought to be the most important proapoptotic protein [6,7]. In normal conditions, Bax
is mainly localized in the cytosol. Upon apoptotic stimuli, Bax undergoes a conformational change and translocates to the outer mitochondrial membrane [8]. Then, Bax becomes oligomerized and permeabilizes the mitochondrial membrane to release apoptotic factors [29,30]. Currently, there is a great interest in understanding how the Bax activity is regulated during apoptosis. It has been shown that several proteins, including Mcl-1, Ku70, 14-3-3 and Humanin, sequester Bax in the cytoplasm [31–35]. Several anti-apoptotic Bcl-2
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family proteins, such as Bcl-2, Bcl-XL in the mitochondria, also inhibit Bax translocation and activation [36,37]. However, under certain apoptotic stimuli, Bax can be activated directly by certain BH3-only proteins (e.g. tBid and Bim) and undergoes a conformational change [14,16,17,38,39]. In this study, we use a yeast two-hybrid assay to identify RACK1 as a novel binding partner of Bax. We confirmed that RACK1 interacts with Bax in mammalian cells and found that RACK1 promotes Bax oligomerization. Our study provided a new mechanism for the regulations of Bax oligomerization. The balance between pro- and anti-apoptotic proteins helps determine the susceptibility of cells in response to a cell death signal. Bax with Bcl-XL are two key pro- and anti-apoptotic proteins. Bcl-XL is known to interact with Bax and inhibit Bax oligomerization during apoptosis [9]. In this study, we observed that the interaction of Bax with Bcl-XL is interrupted by overexpressed RACK1, and the interaction of Bax with Bcl-XL is enhanced in cells in depletion of RACK1 (Fig.3C and D). These results indicated that RACK1 inhibits the interaction of Bax and Bcl-XL. In an apoptosis experiment, we observed that RACK1-overexpressing cells undergo apoptosis while depleting RACK1 reduces UV-induced apoptosis. Therefore, we proposed that RACK1 functions in Bax oligomerization to induce apoptosis. RACK1 has been reported as an E3 ligase component to mediate the degradation of DeltaNp63alpha, a member of the p53 family [40] and to interact with Elongin C and mediate the degradation of HIF-1α [41]. In our experiments, we did not observe any effect of protein degradation of Bax and Bcl-XL under RACK1 overexpression (data not shown). We explain that RACK1 is not an E3 ligase although it can participate in the formation of an E3 ligase complex. When it functions in forming an E3 ligase complex, RACK1 may require an E3 ligase. However, in the situation of Bax, RACK1 cannot recruit any E3 ligase. Therefore RACK1 could not mediate the degradation of Bax. Our result that RACK1 promotes Bax oligomerization to induce apoptosis in Hela cells is consistent with the observations in the HT-29 cells [24]. Especially, our observation that RACK1 promotes the oligomerizatioin of Bax examined by immunostaining experiment is almost the same as the observation from a previous report (comparing the Fig. 3B in literature [24] with our results in Fig. 2B). In this study, we have provided further evidence that RACK1 interacts with Bax and promotes the oligomerization of Bax by immunoprecipitation experiment. Manidipudi V and Cartwright CA analyzed that RACK1 inhibits the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL and induces the expression of proapoptotic protein Bim [24]. Our study provides another mechanism that RACK1 promotes apoptosis by disassociating the interaction of Bax with Bcl-XL, which is similar to the mechanism by which p53 regulates the activity of Bax [15]. However, other groups reported that RACK1 inhibits apoptosis [21–23]. These controversial observations demonstrated a diverse role of RACK1 in different tumors with different target proteins. Indeed, RACK1 contains 7 WD domains which mediate the association of different substrates. We believe that the roles of RACK1 on cell survival or death are dependent on the dominance of different targeted proteins. Further study is required to reveal the detailed functions of RACK1 in different cells. 4. Materials and methods 4.1. Plasmids and reagents pcDNA3.1/Myc-RACK1 and pGEX4T-2/GST-RACK1 were generated by inserting the PCR-amplified related inserts into pcDNA3.1/Myc and pGEX4T-2. pcDNA3.1/Myc-Bax and pcDNA3.1/Flag-Bax were generated by inserting the PCR-amplified related inserts into a pcDNA3.1/ Myc and pcDNA3.1/Flag. Constructs expressing GFP-Bax, YFP-ART, YFP-ART-BH3, YFP-BH3, YFP-α5/6, YFP-α5/6-TM, YFP-TM, and GFPBcl-XL were kindly provided by Dr. Donald Chang, Hong Kong University of Science & Technology, Hong Kong, China. pcDNA3.1/
Myc, pcDNA3.1/Flag and pGEX4T-2 were kept in this lab. Antibody against Flag (M2) was from Sigma. Antibodies against Myc (9E10) and GFP were purchased from Santa Cruz. Fluorescent secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. The target sequences of siRNA are from 146 to 166 bp (human RACK1 sequence accession no. NM_006098), which is confirmed by Cartwright [42]. 4.2. Cell culture HEK293T and Hela cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. All the cells were kept at 37 °C in 5% CO2-containing atmosphere. The medium and serum were purchased from Gibco. For UV treatment, medium was removed and saved, cells were rinsed with PBS and irradiated, and medium was restored. Cells were exposed to UV irradiation at a fluence of 120 mJ/cm2 and observed at 5 h after treatment. 4.3. Co-immunoprecipitations HEK293T cells were plated in 60 mm dishes the day before transfection. 5 μg of pcDNA3.1/Myc-RACK1 and the indicated plasmids were transfected. If necessary, pcDNA3.1/Myc or pcDNA3.1/Flag empty vector was added to ensure that an equal amount of plasmid was transfected each dish. After transfection for 18-36 h, cells were lysed in 600 μl cell lysis buffer (50 mM Tris·Cl, 150 mM NaCl, 50 mM NaF, 0.5% NP-40, pH 7.5) with protease inhibitors to prepare wholecell lysates. The lysate was mixed with proper antibodies and incubated at 4 °C for 2 h, followed by addition of protein G-agarose beads to pellet the immune complex. The immunoprecipitants and 5% of lysates were analyzed by immunoblotting for the indicated proteins. 4.4. GST pull down assays GST-fused RACK1 (GST-RACK1) was expressed in E. coli strain BL21 cells and purified by affinity chromatography with glutathioneSepharose 4B resin (GE Healthcare). 50 μg of each purified protein was mixed with glutathione-Sepharose beads and incubated at 4 °C for 30 min before use. Constructs expressing the indicated plasmids were tansfected into HEK293T cells individually. After transfection for 18–36 h, the whole-cell lysates were prepared and incubated with appropriate GST fusion protein/glutathione-Sepharose beads complex at 4 °C for 2 h. The precipitates were analyzed by immunoblotting for the indicated proteins. 4.5. Immunofluorescent staining and microscopy Hela cells were cultured on 6-well plates with 8 × 104 cells per well (Corning Incorporated, Corning, NY). Cells were transfected with the indicated plasmids and fixed after 36 h transfection with 4% paraformaldehyde for 20 min and perforated with 0.3% Triton X-100 for 10 min. The cells were blocked with 10% FBS for 50 min followed by incubation with c-Myc antibody at 4 °C overnight. The cells were incubated with a secondary antibody conjugated with goat antimouse IgG/TRITC antibody (Jackson Research Laboratories) for 1 h and counterstained with DAPI for 10 min. Finally, the results were visualized by a confocal laser scanning microscope (OLYMPUS BX61). The co-localization of the two proteins was shown as a merged image. 4.6. Flow cytometry To quantify cells in early and late apoptosis, transfectants were incubated with annexin V–FITC/PI for 10–15 min at RT, according to the manufacturer's protocol (Pharmingen) and analyzed by flow cytometry. Cells (104) were analyzed by an FACScan cell sorter (Becton-Dickinson,
Y. Wu et al. / Cellular Signalling 22 (2010) 1495–1501
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