www.nature.com/scientificreports opeN Received: 15 January 2016 Accepted: 11 July 2016 Published: 08 August 2016 Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2 Rosi Bissinger1, elisabeth Lang2, Mehrdad Ghashghaeinia1, Yogesh singh1, Christine Zelenak3, Birgit Fehrenbacher4, sabina Honisch1, Hong Chen1, Hajar Fakhri1, Anja t. Umbach1, Guilai Liu1, Rexhep Rexhepaj1,5, Guoxing Liu1, Martin schaller4, Andreas F. Mack6, Adrian Lupescu1, Lutz Birnbaumer7, Florian Lang1 & syed M. Qadri1,8,9 putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (ps) exposure. eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. spontaneous ps exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death. G protein-coupled receptors activate heterotrimeric G proteins via ligand binding, thereby modulating the activity of cellular effectors and consequently regulating a wide array of cell functions1,2. The putative function of the functional class of G protein Gαi is defined by their ability to downregulate cAMP levels by inhibition of adenylyl cyclase2,3. The closely-related Gα members Gαi1, Gαi2, and Gαi3, sharing 85–95% of their amino acid sequence identity, are characterized by their sensitivity to pertussis toxin2,3. Gαi2, the quantitatively predominant Gαi isoform, is a decisive regulator of leukocyte, endothelial and platelet functions4–7. Further putative roles of Gαi2 signaling include ion channel regulation, cell differentiation, proliferation and apoptosis8–12. Effector kinases of G-protein signaling include phosphoinositide 3-kinases13, which are known to be involved in the regulation of apoptosis14. Gαi2 further influences Ca2+ signaling in nucleated cells by the activation of TRPC4 channels which, in turn, increases Ca2+ influx15. In cardiomyocytes, Gαi2 has been shown to modulate the activity of L-type 1 institute of cardiology, Vascular Medicine and Physiology, University of tuebingen, Germany. 2Department of Gastroenterology, Hepatology and infectious Diseases, University of Duesseldorf, Germany. 3Department of internal Medicine, charité Medical University, Berlin, Germany. 4Department of Dermatology, University of tuebingen, Germany. 5institute of Biochemistry and Molecular Biology, University of Bonn, Germany. 6institute of Anatomy, University of tuebingen, Germany. 7neurobiology Laboratory, national institute of environmental Health Sciences, national institutes of Health, Research triangle Park, Durham, nc, USA. 8institute of Biomedical Research (BiOMeD), School of Medical Sciences, catholic University of Argentina, Buenos Aires, Argentina. 9Department of Pathology and Molecular Medicine, McMaster University, Hamilton, On, canada. correspondence and requests for materials should be addressed to F.L. (email: florian.lang@uni-tuebingen.de) Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 1 www.nature.com/scientificreports/ Figure 1. Blood parameters. Means ± SEM of erythrocyte count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and reticulocyte count (RTC) (A, n = 8), Ter119/CD71 positive cells (C, n = 6), plasma erythropoietin (EPO) levels (D, n = 3–4), leukocyte count (E, n = 8) and platelet count (F, n = 8) in Gαi2+/+ and Gαi2−/− mice. ***(p < 0.001) significantly different from Gαi2+/+ mice. (B) May-Grünwald staining of erythrocytes from Gαi2+/+ and Gαi2−/− mice. voltage-dependent Ca2+-channels11. Furthermore, Gαi2 is a powerful regulator of cytosolic Ca2+ activity in islet beta cells12 and neutrophils16, thus, regulating a variety of Ca2+-dependent cell functions. Phenotypically, Gαi2 knockout mice have been reported to display a predisposition towards a wide range of disorders including growth retardation, inflammatory bowel disease, carcinogenesis, cardiac arrhythmia and impaired haemostasis4,17,18. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis19,20, which, similar to apoptosis, is triggered by osmotic shock and characterized by cell shrinkage and cell membrane scrambling20,21. Eryptosis may be triggered by activation of Ca2+-permeable cation channels20 which subsequently leads to increase of cytosolic Ca2+. The molecular identity of these cation channels has not been completely characterized but apparently involves TRPC6 channels22. The cation channels are activated by prostaglandin E2, which is formed following exposure of erythrocytes to hyperosmotic shock19. The channels are further activated by a wide variety of cell stressors, xenobiotics and endogenous mediators19. Ca2+ activates Ca2+-sensitive K+ channels with exit of KCl and osmotically obliged water and thus cell shrinkage19,20. An increase of cytosolic Ca2+ is further followed by stimulation of cell membrane scrambling with exposure of phosphatidylserine (PS) at the cell surface19,20. The Ca2+ sensitivity of cell membrane scrambling is further enhanced by ceramide21. PS-exposing cells are bound to macrophages, engulfed and degraded and thus cleared from circulating blood19,20,23–25. To the best of our knowledge, the impact of Gαi2 on erythrocyte survival and suicidal death has hitherto not been reported. In the present study we explored whether the Gαi2 isoform is expressed in erythrocytes and whether it participates in the regulation of erythrocyte survival. To this end, eryptosis was determined in erythrocytes from Gαi2 knockout mice (Gαi2−/−) and their wild type littermates (Gαi2+/+). Results The present study addressed the impact of Gαi2 on eryptosis in mice. To this end, experiments were performed in mice lacking functional Gαi2 (Gαi2−/−) and corresponding wild type mice (Gαi2+/+). As shown in Fig. 1A, erythrocyte count, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin, mean corpuscular Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 2 www.nature.com/scientificreports/ Figure 2. Gαi2 expression. (A) Western blots showing Gαi2 (40 kDa) and GAPDH (37 kDa) expression in erythrocytes from Gαi2+/+ (bands 1, 2 and 3) or Gαi2−/− (bands 5, 6 and 7) mice and humans (band 4) in whole blood (bands 1 and 5), diluted whole blood (bands 2 and 6; 1:3.7 dilution) and purified erythrocytes (bands 3 and 7). (B) Means ± SEM of Gαi2 abundance in murine and human erythrocytes relative to the loading control GAPDH (n = 3). hemoglobin concentration and the percentage of reticulocytes were not significantly different between Gαi2−/− and Gαi2+/+ mice. The mean corpuscular volume was slightly, but significantly larger in Gαi2−/− than in Gαi2+/+ erythrocytes (41.1 ± 0.3 fl for Gαi2+/+ mice versus 42.8 ± 0.2 fl for Gαi2−/− mice; n = 8, p < 0.001). Gαi2−/− erythrocytes are, thus, normochromic and moderately larger as compared to Gαi2+/+ erythrocytes. May-Grünwald staining further revealed no apparent changes in erythrocyte shape from Gαi2−/− mice as compared to erythrocytes from Gαi2+/+ mice (Fig. 1B). The percentages of CD71/Ter119 positive cells were similar in Gαi2−/− and Gαi2+/+ mice suggesting similar patterns of dynamic erythrocyte maturation in vivo (Fig. 1C). Plasma erythropoietin concentrations were further similar in Gαi2−/− and Gαi2+/+ mice (Fig. 1D). Consistent with a previous report26, we observed leukocytosis in Gαi2−/− mice (Fig. 1E) which is attributed to increased production of proinflammatory cytokines in Gαi2−/− mice18. The platelet count in Gαi2−/− mice was, however, not significantly different from Gαi2+/+ mice (Fig. 1F). Immunoblotting was employed to examine whether Gαi2 is expressed in human and murine erythrocytes. To this end, erythrocytes from humans or from mice were isolated and purified. Equal amounts of protein lysates were immunoblotted. GAPDH served as a loading control. As depicted in Fig. 2A, the incubation with Gα i2 specific antibodies yielded a single band of 40 kDa in human erythrocytes as well as erythrocytes from Gαi2+/+ mice, but not in erythrocytes from Gαi2−/− mice. The bands appearing below 40 kDa are presumably the result of non-specific antibody binding. Densitometry analysis revealed that Gαi2 protein is significantly more abundant in mouse erythrocytes as compared to human erythrocytes (Fig. 2B). Thus, Gαi2 is expressed in both human and murine erythrocytes. Next, we explored whether Gαi2 deficiency influences erythrocyte survival. To this end, using annexin V binding, forward scatter and Fluo3 fluorescence in FACS analysis we analyzed erythrocyte cell membrane PS exposure, cell shrinkage and cytosolic Ca2+ activity, respectively. As depicted in Fig. 3A, freshly drawn and untreated erythrocytes were visualized using confocal microscopy and quantification of multiple fields showed a decreased ratio of annexin V binding cells to total cells (observed under transmission light) per field in Gαi2−/− erythrocytes (0.028 ± 0.007; n = 4) as compared to Gαi2+/+ erythrocytes (0.069 ± 0.007; n = 4). PS exposure was simultaneously quantified using FACS analysis (50,000 cells were quantified) and confirmed that in both freshly drawn blood (Fig. 3B,C) and following 12 h incubation in Ringer solution (Fig. 3D), the percentage of annexin V binding erythrocytes was significantly lower in Gαi2−/− mice than in Gαi2+/+ mice. Quantification of forward scatter showed that the cell volume was significantly larger in Gαi2−/− erythrocytes as compared to Gαi2+/+ erythrocytes (Fig. 4A,B). Both cell membrane PS exposure and cell shrinkage are influenced by cytosolic Ca2+ activity20. As shown in Fig. 4C,D, the percentage of Fluo3 positive erythrocytes was slightly but significantly lower in Gαi2−/− mice as compared to Gαi2+/+ mice. These data suggest an inhibitory effect of Gαi2 deficiency on eryptosis. Further experiments then addressed the susceptibility of Gα i2-deficient erythrocytes to osmotic shock ex vivo, a pathophysiological cell stressor and a known stimulator of eryptosis. As illustrated in Fig. 5A,B, exposure of erythrocytes for 30 min to hyperosmotic Ringer (550 mM sucrose was added to the Ringer solution to reach the final osmolarity of 850 mOsm), significantly enhanced PS exposure, an effect, however, significantly blunted in Gαi2−/− erythrocytes as compared to Gαi2+/+ erythrocytes. Erythrocyte forward scatter was quantified to determine hyperosmotic shock-triggered cell shrinkage. As shown in Fig. 5C,D, forward scatter was significantly reduced by hyperosmotic shock in erythrocytes from both Gαi2−/− and Gαi2+/+ mice. The effect was significantly less pronounced in Gαi2−/− erythrocytes than in Gαi2+/+ erythrocytes. To elucidate the mechanism contributing to the protective effect of Gαi2 deficiency against hyperosmotic shock-triggered eryptosis, we determined erythrocyte cytosolic Ca2+ activity following hyperosmotic shock. As shown in Fig. 6A,B, exposure of erythrocytes to hyperosmotic shock significantly enhanced the percentage of Fluo3 positive erythrocytes. The effect was, however, significantly blunted in Gαi2−/− erythrocytes as compared Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 3 www.nature.com/scientificreports/ Figure 3. Phosphatidylserine externalization. (A) Confocal microscopy of annexin-V-fluorescence (right panels) and transmission light (middle and left panels) of erythrocytes from Gαi2+/+ and Gαi2−/− mice. Middle panels are amplified images of the area inside the squares of left panels. (B) Histogram (Blue: Gαi2+/+, red: Gαi2−/−) and means ± SEM of annexin-V-binding in erythrocytes freshly drawn (C, n = 24–40) or incubated 12 h in Ringer (D, n = 11–17). *(p < 0.05) significantly different from Gαi2+/+ mice. to Gαi2+/+ erythrocytes. Further experiments explored the resistance of erythrocytes to a decline of extracellular osmolarity. As illustrated in Fig. 6C, the resistance of erythrocytes to graded decrease of osmolarity was significantly lower in Gαi2+/+ than in Gαi2−/− erythrocytes. Thus, Gαi2 deficiency counteracts the sensitivity of erythrocytes to both hyper- and hypoosmotic shock. Additional experiments explored whether erythrocyte Gαi2 deficiency protects against ceramide-sensitive eryptosis. As shown in Fig. 7, treatment of erythrocytes from Gαi2−/− and Gαi2+/+ mice with C6 ceramide and bacterial sphingomyelinase significantly increased PS exposure, an effect, slightly, but significantly less pronounced in Gαi2−/− erythrocytes as compared to Gαi2+/+ erythrocytes. Thus, erythrocyte Gαi2 deficiency has a subtle effect on ceramide-elicited eryptosis. Discussion The present observations disclose the expression of Gαi2 in human and murine erythrocytes and further reveals that Gαi2 deficiency confers partial protection against suicidal erythrocyte death or eryptosis. Our findings show that the percentage of eryptotic cells in circulating blood is significantly lower in Gαi2−/− mice as in Gαi2+/+ mice. Gαi2−/− mice do not show overt changes in erythrocyte parameters such as erythrocyte count, hematocrit, hemoglobin concentration and reticulocyte count. The impact of Gαi2 deficiency on erythrocytes is unmasked in the presence of pathophysiological cell stressors ex vivo such as hyperosmotic shock and following treatment with C6 ceramide and bacterial sphingomyelinase, whereby eryptosis is significantly less pronounced in Gαi2−/− erythrocytes as compared to Gαi2+/+ erythrocytes. Our data show that in the absence of stress, the difference between the percentage of PS-exposing erythrocytes in Gαi2+/+ mice and Gαi2−/− mice is subtle (~0.2%) yet statistically significant. Previous studies have shown that spontaneous PS exposure in freshly drawn erythrocytes from healthy wild-type mice of different strains does not exceed 1%19 of the total number of circulating erythrocytes. Thus, in transgenic mice which display a phenotype of reduced eryptosis, the percentage of PS-exposing circulating erythrocytes may be significantly lower than in wild-type mice despite relatively lower magnitudes of difference. Exposure of erythrocytes to hypertonic extracellular environment in vitro simulates the osmotic conditions encountered in the kidney medulla20. In conditions Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 4 www.nature.com/scientificreports/ Figure 4. Cell shrinkage and cytosolic Ca2+-activity. Histogram (A,C; Blue: Gαi2+/+, red: Gαi2−/−) and means ± SEM of forward scatter (B, n = 21–33) and percentage of Fluo3 positive erythrocytes (D, n = 8–16). *,**(p < 0.05, p < 0.01) significantly different from Gαi2+/+ mice. such as acute renal failure, erythrocytes may enter eryptosis due to their entrapment in the kidney medulla21. Gα i2 deficiency may blunt eryptosis and thus favorably influence the respective clinical condition. Our data show that, in addition to curtailing PS exposure, Gαi2−/− erythrocytes showed increased resistance to cell shrinkage following hyperosmotic shock. Accordingly, the mean corpuscular cell volume was significantly larger in Gαi2−/− erythrocytes. Along those lines, it is intriguing to speculate that Gαi2 influences cell volume regulatory ion channels in erythrocytes. Mechanistically, hyperosmotic shock is a powerful stimulator of Ca2+ entry and ceramide formation in erythrocytes20. We observed that following hyperosmotic shock of erythrocytes, Gαi2 deficiency leads to subtle but significant decrease of cytosolic Ca2+ entry. On the other hand, Gαi2 may additionally mediate hyperosmotic shock-induced eryptosis by influencing ceramide signaling21. This is corroborated by our data showing a mitigating effect of Gαi2 deficiency on eryptosis triggered by either C6 ceramide or bacterial sphingomyelinase. Ceramide sensitizes erythrocytes to the eryptotic effect of enhanced Ca2+ concentration and may stimulate eryptosis without appreciable increase in cytosolic Ca2+ activity27. Ceramide further modifies the interaction of the erythrocyte membrane with the cytoskeleton thereby increasing membrane fragility28. As Gαi2 is an essential regulator for Ca2+ signaling in nucleated cells, it is possible that the inhibitory effect of Gαi2 deficiency on erythrocyte death is, at least in part, mediated by its influence on cytosolic Ca2+ activity. Eryptosis is inhibited by catecholamines including dopamine29. Interestingly, dopamine-dependent signaling involves pertussis toxin-sensitive Gαi230. Further signaling molecules that regulate the eryptosis machinery include AMPK20, p38 MAPK31, CK1α32, PAK233, PDK120, MSK1/234 and CDK435. Eryptosis is triggered by a myriad of xenobiotics and endogenous substances20,36–48, and accelerated eryptosis contributes to the anemia associated with several clinical disorders20, including iron deficiency49, sepsis50, renal failure51, hepatic failure52, malignancy24, ageing53 and Wilson’s disease54. Eryptotic erythrocytes adhere to the vascular wall55, and stimulate blood clotting56. Excessive eryptosis may thus interfere with microcirculation and participate in the vascular injury of metabolic syndrome57. Accordingly, Gαi2−/− mice may be particularly resistant to derangements of microcirculation following exposure to triggers of eryptosis. Moreover, eryptosis has been shown to influence the quality of stored erythrocytes58. Pharmacologically targeting Gαi2, at least in theory, may further provide new avenues in the treatment of conditions associated with anemia resulting from increased eryptosis20. On the other hand, Gαi2 modulation may serve as a novel target for the treatment of malaria, a condition where eryptosis plays a protective role in ameliorating parasitemia by expediting the clearance of pathogen-infected erythrocytes20. Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 5 www.nature.com/scientificreports/ Figure 5. Effect of hyperosmolarity on phosphatidylserine externalization and cell shrinkage. Histogram (A,C; Blue: Gαi2+/+, red: Gαi2−/−) and means ± SEM of annexin-V-binding (B, n = 11–14) and forward scatter (D, n = 11–14) following 30 min incubation in isosmotic (300 mOsm) or hyperosmotic (850 m Osm) Ringer. ### (p < 0.001) significantly different from isosmotic, *,**,***(p < 0.05, p < 0.01, p < 0.001) from Gαi2+/+. In conclusion, the G-protein subunit Gαi2 is expressed in human and murine erythrocytes and participates in the regulation of erythrocyte suicide. Materials and Methods Experiments were performed in Gα i2 knockout mice (Gαi2−/−) and their wild type littermates (Gαi2 ) of 6–9 weeks of age. The mice were generated and initially characterized on a SV129 background18. Mice were backcrossed on a C57BL6 background and kept under specified pathogen-free (SPF) environment in individually ventilated cages (IVC) to prolong life expectancy4,59. All animal experiments were conducted according to the German law for the care and use of laboratory animals and were approved by local government authorities (Regierungspräsidium Tübingen). Mice. +/+ Blood count, incubation and solutions. For all experiments except for the blood count, heparin blood was retrieved from the retrobulbar plexus of mice. For the blood count, EDTA blood was analyzed using an electronic hematology particle counter (type MDM 905 from Medical Diagnostics Marx; Butzbach, Germany) equipped with a photometric unit for haemoglobin determination. Plasma erythropoietin levels were determined using an immunoassay kit according to the manufacturer’s instructions (R&D Systems, Wiesbaden, Germany). Murine erythrocytes were isolated by being washed two times with Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1 MgSO4, and 32 HEPES/NaOH (pH 7.4), 5 glucose, and 1 CaCl2. Where indicated, sucrose (550 mM), C6 ceramide (50 µM; Sigma) or bacterial sphingomyelinase (0.01 U/ml; Sigma) were added to the Ringer solution. May-Grünwald staining was used to examine changes in erythrocyte shape. Briefly, 20 µl of erythrocytes were smeared and fixed using methanol onto a glass slide, and stained with 5% Giemsa Azur-Eosin (Merck Millipore, Germany) in phosphate-buffered saline (in mM: 1.05 KH2PO4, 2.97 Na2HPO4, 155.2 NaCl) for 20 min. Subsequently, images were taken on a Nikon Diaphot 300 Microscope (Nikon Instruments, Germany). Reticulocyte count and markers of erythrocyte maturation. For determination of the reticulocyte count EDTA-whole blood (2.5 µl) was added to 500 µl Retic-COUNT (Thiazole orange) reagent from Becton Dickinson. Samples were stained for 30 min at room temperature, and flow cytometry was performed according to the manufacturer’s instructions. Forward scatter (FSC), side scatter (SSC), and thiazole orange-fluorescence intensity Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 6 www.nature.com/scientificreports/ Figure 6. Effect of osmotic changes on cytosolic Ca2+-activity and hemolysis. Histogram (A; Blue: Gαi2+/+, red: Gαi2−/−) and means ± SEM of the percentage of erythrocytes with enhanced Fluo3-fluorescence (B, n = 11–14) following 30 min incubation in isosmotic (300 mOsm) or hyperosmotic (850 mOsm) Ringer. (C) Means ± SEM (n = 3–5) of relative hemolysis as a function of extracellular osmolarity (in % of isomotic Ringer) in Gαi2+/+ (blue) and Gαi2−/− (red) erythrocytes. ###(p < 0.001) significantly different from isosmotic, *(p < 0.05) from Gαi2+/+. (in FL-1) of the blood cells were determined. The number of Retic-COUNT positive reticulocytes was expressed as the percentage of the total gated erythrocyte populations. Gating of erythrocytes was achieved by analysis of FSC vs. SSC dot plots using CellQuest software. To further examine the dynamic maturation of erythrocytes in vivo, erythrocytes were double stained with CD71 (1:12.5; BD Biosciences), and Ter119 (1:250; BD Biosciences). Ter119 and CD71 positive cells were quantified by analyzing the upper right quadrant of an FL1 versus FL2 dot plot. phosphatidylserine exposure and forward scatter. After incubation, erythrocytes were washed once in Ringer solution containing 5 mM CaCl2. The cells were then stained with annexin-V-FITC (1:250 dilution; Immunotools, Friesoythe, Germany) at a 1:500 dilution. After 15 min, samples were measured by flow cytometric analysis (FACS-Calibur; BD). Cells were analyzed by forward scatter, and annexin V fluorescence intensity was measured in fluorescence channel FL-1 with excitation and emission wavelengths of 488 nm and 530 nm, respectively, on a FACS Calibur (BD, Heidelberg, Germany) as described previously24. Where indicated, spontaneous PS exposure and forward scatter were determined by addition of 2 µl of freshly drawn erythrocytes in 500 µl Ringer solution containing 5 mM CaCl2 and annexin-V-FITC. Raw data for annexin V positive erythrocytes was collected by a primary gating of the erythrocyte population on FSC vs. SSC dot plots and, subsequently, by setting an arbitrary marker at the base of the cell population on an FL1 histogram. The cell population plotted on the left of the arbitrary marker was considered positive for annexin V binding. estimation of intracellular Ca2+. For measurement of intracellular Ca2+, 50 µl erythrocyte suspension was washed in Ringer solution and then loaded with Fluo-3/AM (Biotrend, Köln, Germany) in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µl Ringer. Then, Ca2+-dependent fluorescence intensity was measured in the fluorescence channel FL-1 in FACS analysis. Where indicated, spontaneous intracellular Ca2+ was determined by addition of 2 µl of freshly drawn erythrocytes in 500 µl Ringer solution containing 5 mM CaCl2 as well as Fluo3/AM. Fluo3 positive cells were plotted using an FL1 histogram similar to the analysis of annexin V positive cells. Determination of the osmotic resistance. Two microliters of blood were added to 200 µl of PBS solutions with decreasing osmolarity. After centrifugation for 5 min at 3000 rpm, the supernatant was transferred to a Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 7 www.nature.com/scientificreports/ Figure 7. Effect of C6-ceramide and bacterial sphingomyelinase on phosphatidylserine externalization. Histograms (A,C; Blue: Gαi2+/+, red: Gαi2−/−) and means ± SEM of annexin-V-binding following exposure to C6-ceramide (A,B; 50 µM, 12 h; n = 11–17) or bacterial sphingomyelinase (C,D; 0.01 U/ml 24 h; n = 7–16). ### (p < 0.001) significantly different from Control. *(p < 0.05) from Gαi2+/+. 96-well plate, and the absorption at 405 nm was determined as a measure of hemolysis. Absorption of the supernatant of erythrocytes lysed in pure distilled water was defined as 100% hemolysis. Immunoblotting. To examine the expression of Gαi2 in human or murine erythrocytes, 150 µl erythrocyte pellet was lysed in 50 ml of 20 mM HEPES/NaOH (pH 7.4). Ghost membranes were pelleted (15,000 g for 20 min at 4 °C) and lysed in 200 µl lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; 0.5% SDS; 1 mM NaF; 1 mM Na3VO4; and 0.4% β-mercaptoethanol) containing protease inhibitor cocktail (Sigma, Schnelldorf, Germany). Triton X-100, a non-ionic detergent, was used in erythrocyte ghost preparation due to its effective solubilization power and a relatively mild effect on membrane-bound enzymes60. In all cases, 60 µg of protein was solubilized in Laemmli sample buffer at 95 °C for 5 min and resolved by pre-casted 10% SDS-PAGE gel (Invitrogen, Karlsruhe, Germany). For immunoblotting, proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane and blocked with 5% nonfat milk in TBS-0.10% Tween 20 at room temperature for 1 h. Then, the membrane was incubated with anti-G-protein alpha inhibitor 2 antibody (1:5000; 40 kDa; Abcam Cat# ab157204) at 4 °C overnight. After being washed (in TBS-0.10% Tween 20) and subsequently blocked, the blots were incubated with secondary anti-rabbit antibody (1:2000; Cell Signaling) for 1 h at room temperature. After being washed, the antibody binding was detected with the ECL detection reagent (Amersham, Freiburg, Germany). Confocal microscopy and immunofluorescence. For the visualization of eryptotic erythrocytes, 4 µl of erythrocytes, incubated in the respective experimental solutions, were stained with FITC-conjugated annexin-V (1:100 dilution; ImmunoTools, Friesoythe, Germany) in 200 µl Ringer solution containing 5 mM CaCl2. Then, the erythrocytes were washed twice and finally resuspended in 50 µl of Ringer solution containing 5 mM CaCl2. Twenty µl were mounted with Prolong Gold antifade reagent (Invitrogen, Darmstadt, Germany) onto a glass slide and covered with a coverslip. Sections were analyzed using a Leica TCS-SP / Leica DM RB confocal laser scanning microscope. Images were processed with Leica Confocal Software LCS (Version 2.61). statistics. Data are expressed as arithmetic means ± SEM, and statistical analysis was made using ANOVA or t-test, as appropriate. n denotes the number of different erythrocyte specimens studied. 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Acknowledgements The authors thank Prof. Bernd Nürnberg and members of his laboratory (Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Germany) for their valuable suggestions and for providing blood from Gαi2−/− mice. The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch and the technical assistance of Efi Faber and Annette Knoblich. This study was supported by Deutsche Forschungsgemeinschaft (Nr. La 315/13-3) and the Intramural Research Program of the NIH (project Z01-ES-101643 to LB). Author Contributions F.L. and S.M.Q. designed the project and wrote the main manuscript text. R.B., E.L., M.G., Y.S., C.Z., B.F., S.H., H.C., H.F., A.T.U., G.L., R.R., G.L., M.S., A.F.M., A.L., L.B. and S.M.Q. performed the acquisition, analysis and/or interpretation of data. R.B., M.G., Y.S., B.F. and S.M.Q. prepared the figures. All authors have read and reviewed the manuscript and approved the final version. Additional Information Competing financial interests: The authors declare no competing financial interests. How to cite this article: Bissinger, R. et al. Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2. Sci. Rep. 6, 30925; doi: 10.1038/srep30925 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific RepoRts | 6:30925 | DOI: 10.1038/srep30925 10