The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R−/− mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R Mycobacterium tuberculosis or Mycobacterium bovis. Two hypervirulent strains (Mtb strain 1471 and Mbv strain MP287/03) induced extensive pulmonary inflammation and necrosis in mice and promoted bacillus dissemination and animal death. We hypothesized that the innate immune response to endogenous damage signals from necrotic cells could aggravate the disease. We focused our study on the purinergic P2X7 receptor (P2X7R), a sensor of ATP that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. Our data provide new insights into the pathogenesis of severe tuberculosis by showing that mice that lack P2X7R have attenuated disease with substantially reduced bacillus dissemination and lung inflammation without evidence of necrosis.Nearly 9 million new cases of tuberculosis and 1.3 million deaths are reported yearly worldwide. Most individuals infected with tubercle bacilli remain asymptomatic; however, some develop active tuberculosis due to the reactivation of latent infections. Progressive primary tuberculosis is an alternative form of the disease that mostly affects children and immunocompromised individuals. Extensive pneumonia, pulmonary necrosis and bacillus dissemination characterize some of the aggressive forms of tuberculosis. To investigate the molecular mechanisms that underlie severe disease progression, we used experimental models of relatively resistant C57BL/6 mice that were infected with highly virulent strains of  Mycobacterium tuberculosis complex (Mycobacterium tuberculosis – Mtb and Mycobacterium bovis – Mbv), although most effectively control the pathogen and remain asymptomatic by establishing equilibrium with the bacilli Tuberculosis (TB) is a serious public health issue, with nearly 9 million new annual cases of the disease worldwide and 1.3 million deaths per year The reactivation of latent infections in immunocompetent individuals occurs at rates that range from 3% to 10% per lifespan Mouse TB models have been extremely useful in the characterization of mechanisms that are involved in disease pathogenesis and the generation of protective immunity. However, it is difficult to determine the factors that are responsible for the development of pulmonary necrosis because this process is atypical in mice that are infected with virulent mycobacteria, such as the H37Rv reference strain Mtb strains belonging to the emerging Beijing genotype family, which are associated with high virulence, transmissibility and drug resistance Mtb genotypes, promote unusual rapid fatal diseases in infected mice that are characterized by a severe pulmonary pathology, with high bacterial burden and extensive lung consolidation and areas of focal necrosis Mbv strains are able to cause sudden pneumonia with extensive necrosis that lead to early death Recent epidemiologic studies have shown that in vivo to concentrations greater than 100 µM during pathological processes Necrotic cell death permits the release of damage-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP). Extracellular ATP (eATP) is a danger signal for cells of the immune system and contributes to inflammatory and reparatory responses Mtb strain 1471 (Beijing genotype) and Mbv strain MP287/03. These mycobacterial strains were epidemiologically associated with outbreaks of TB in human and bovine populations, respectively, and showed high virulence in our previous in vitro studies −/− and C57BL/6 mice. Our data provide new insights into the pathogenesis of severe TB by showing a requirement for P2X7R in the induction of macrophage death and the progression of tuberculous pneumonia, necrotic pulmonary lesions, bacillus dissemination and anticipated animal death.Based on these findings, we were determined to investigate whether the recognition of eATP from necrotic cells by P2X7R could elicit an innate immune response and contribute to the development of severe TB. To investigate this possibility, we used murine models of pulmonary TB induced by intratracheal infections with the highly virulent isolates Mtb and Mbv strains were selected from 9 strains , 1777 (genotype LAM) and H37Rv; Mbv – AN5, B2, B29, MP287/03 and MP389/03) on the basis of the ability to induce severe TB, i.e., intense pulmonary infiltration with lung necrosis and precocious death of C57BL/6 mice that were infected intratracheally (i.t.) with low doses of bacilli (approx. 100 forms). Beijing 1471 Mtb and MP287/03 Mbv exhibited higher virulence within each subgroup (Mtb and Mbv) compared to the other strains (data not shown). The diseases induced by these strains in C57BL/6 and P2X7R−/− mice were compared to those induced by the virulent H37Rv Mtb strain, which did not induce pulmonary necrosis in C57BL/6 mice. A previous study in P2X7R−/− mice reported no involvement of the P2X7R during pulmonary H37Rv Mtb infection Because ATP released from necrotic cells activates P2X7R and subsequently the innate immune response Mtb killed 90% of C57BL/6 mice until 250 days post-infection (p.i.), whereas MP287/03 Mbv induced 100% mortality before day 40 p.i.  were approximately 10-fold and 1,000-fold higher, respectively, than those in mice infected with H37Rv Mtb (CFU Log10 5.8±0.4)  . Further−/− mice on day 28 p.i. with the Beijing 1471 Mtb, MP287/03 Mbv and H37Rv Mtb strains. Macroscopically, the lungs had white nodules that were more prominent in C57BL/6 mice infected with hypervirulent mycobacteria   and lymphoid  subsets were found in similar proportions in Beijing 1471 Mtb-infected C57BL/6 mice. In contrast, a strong predominance of CD11b+ cells with virtually no T and B cells was observed in MP287/03 Mbv-infected C57BL/6 mice. The absence of P2X7R led to reduced pulmonary infiltration of myeloid cells in both hypervirulent infections and reduced lymphoid cells in the Beijing 1471 Mtb infection. However, MP287/03 Mbv-infected P2X7R−/− mice presented with higher numbers of CD4+ and CD8+ cells compared to their C57BL/6 counterparts. C57BL/6 and P2X7R−/− mice infected with H37Rv Mtb showed comparable numbers of pulmonary myeloid and lymphoid subsets. The CD11b+ population was mostly composed of Ly6C+ cells (monocytes) in all of the infected mice, with the exception of MP287/03 Mbv-infected C57BL/6 mice, which showed a predominance of Ly6G+ cells (neutrophils) .In agreement with the macro- and microscopic analysis of the lungs, C57BL/6 mice infected with Beijing 1471 terparts . For theMtb-infected C57BL/6 mice produced higher levels of IL-1β and IFNγ. In contrast, cells from MP287/03 Mbv-infected mice secreted comparatively lower levels of pro-inflammatory cytokines and higher levels of IL-10, which supports the notion that MP287/03 Mbv is a poor inducer of pro-inflammatory cytokines Mtb infection, cells from P2X7R−/− mice secreted significantly reduced amounts of IL-1β and IFNγ and increased amounts of IL-10 compared to cells from C57BL/6 mice. For the MP287/03 Mbv infection, comparable low levels of IL-1β were found in cell supernatants from C57BL/6 and P2X7R−/− mice, but the lack of P2X7R resulted in an increase of IFNγ and a decrease of IL-10. The H37Rv Mtb infection induced very low production of IL-1β, IFNγ and IL-10; the amount of pro-inflammatory cytokines was significantly reduced in the absence P2X7R.The profiles of cytokines produced by lung infiltrating cells also varied between the two groups of C57BL/6 mice on day 28 p.i. with hypervirulent strains; relatively high or low production levels of pro-inflammatory cytokines were observed during severe TB . More pr−/− mice that were infected with H37Rv Mtb, Beijing 1471 Mtb and MP287/03 Mbv strains. The analysis was performed over a maximum period of 6 days in culture after macrophage differentiation, during which the viability of non-infected BMDMs was maintained above 95%. The intracellular bacterial growth was evaluated at an MOI (multiplicity of infection) of 1, which ensured bacillus multiplication for 6 days p.i. in the absence of spontaneous and bacillus-induced macrophage death. The low MOI was particularly important for hypervirulent mycobacterial strains that grew rapidly and killed the macrophages. The macrophage necrosis induced by the mycobacteria was systematically determined at MOIs of 10 and 20; the best conditions to discriminate C57BL/6 and P2X7R−/− BMDMs are shown.The following experiments were performed to further elucidate the mechanisms that are responsible for the deleterious role of P2X7R in severe TB. The intracellular bacterial growth and dissemination were investigated in bone marrow-derived macrophages (BMDMs) obtained from C57BL/6 and P2X7RMtb and were the most effective strains for inducing cell death  of H37Rv Mtb, Beijing 1471 Mtb and MP287/03 Mbv allowed us to describe the crucial role of P2X7R in the aggressive forms of pulmonary TB. These findings provide a new insight into the pathogenesis of severe TB by showing that mice that lack P2X7R have attenuated disease with substantially reduced bacillus burdens in the lungs, livers and spleens. The absence of P2X7R also promoted significant reductions in pneumonia and lung necrosis on day 28 p.i. with Beijing 1471 Mtb and MP287/03 Mbv, which corroborates our hypothesis of DAMP involvement in the pathogenesis of severe TB. Furthermore, our data are consistent with a previous study in P2X7R−/− mice that reported no role for P2X7R during pulmonary H37Rv Mtb infection The development of two experimental mouse models of human-like severe TB allowed us to determine the general properties of phylogenetically distant strains of hypervirulent mycobacteria that are associated with poor prognosis. The ability of Beijing 1471 +F4/80+Gr1int myeloid cells to the lungs in the exacerbation of H37Rv Mtb infection in mice treated with Poly-IC + myeloid cells with disease attenuation in P2X7R−/− mice that were infected with Beijing 1471 Mtb and MP287/03 Mbv strains. The lack of P2X7R during the Beijing 1471 Mtb infection also resulted in lower IL-1β production by lung infiltrating cells, which could have been responsible for the secretion of less IFNγ and more IL-10. Previous studies showing the resistance of hypervirulent mycobacteria to IFNγ-mediated intracellular killing MtbMtb-infected C57BL/6 mice. Nevertheless, the induction of IL-1β does not fully explain how P2X7R contributes to exacerbate the disease progression during MP287/03 Mbv infection. In this case, severe TB occurred concomitantly with a relatively high secretion of IL-10 and low secretion of IL-1 β and IFNγ, which correlated with low numbers of CD4+ and CD8+ T cells in the lungs. In fact, we have recently shown that MP287/03 Mbv-infected C57BL/6 macrophages are biased towards an M2 immune response profile and induce higher arginase-1 expression and reduced NO production compared to H37Rv Mtb-infected macrophages Mbv infection did not affect the low production of IL-1β by lung infiltrating cells, but it restored the CD4+ and CD8+ T cell responses with the secretion of more IFNγ and less IL-10.P2X7R signaling could detrimentally affect the outcome of severe TB by inducing the production of IL-1β and exacerbating the pulmonary influx of pathogen-permissive monocytes and macrophages. A recent study has implicated the recruitment of CD11bin vitro analysis of C57BL/6 and P2X7R−/− macrophages that were infected with the H37Rv Mtb, Beijing 1471 Mtb and MP287/03 Mbv strains. The abilities of hypervirulent mycobacteria to rapidly grow in C57BL/6 mice and to cause pulmonary necrosis were associated with the abilities to multiply vigorously within C57BL/6 macrophages in vitro and to induce cell death. These findings show a parallel between the in vivo and in vitro behavior of the mycobacterial strains as reported in other studies in vivo data, only Beijing 1471 Mtb elicited the production of high levels of IL-1β in C57BL/6 macrophages, but both hypervirulent mycobacteria strains induced cell death. The subsequent release of live bacilli and the negative staining for annexin V clearly establish that these infected macrophages undergo necrotic cell death. Therefore, with consideration for the critical role of the NLRP3 inflammasome in Mtb-induced necrotic death Mtb and MP287/03 Mbv. Accordingly, the absence of the P2X7R led to a delay in macrophage death that was accompanied by lower release of hypervirulent mycobacteria. This finding indicates that P2X7R cooperates with mycobacterial components exhibiting membrane-lysing activity, such as the ESAT-6 protein from MtbMtb infection in C57BL/6 macrophages −/− macrophages. Thus, even if P2X7R-mediated macrophage death is an evolutionarily selected mechanism of host defense, in Beijing 1471 Mtb and MP287/03 Mbv infections, it allows the bacilli to escape from the innate host defenses and to spread to new cells. A similar behavior was recently described for mycobacteria that possess RD-1 virulence factors, which permit phagolysosomal rupture and allow access to the cytosol An alternative insight into the mechanism responsible for the prejudicial role of P2X7R during severe TB was provided by the Mtb bacilli and the P2X7R-mediated release of viable Beijing 1471 Mtb and MP287/03 Mbv bacilli. The resistance of these hypervirulent mycobacteria to intracellular killing has also been described following macrophage stimulation with IFNγ Mtb infection The deleterious role of P2X7R during severe TB implies that hypervirulent mycobacteria are resistant to the protective mechanisms that are elicited in macrophages following eATP stimulation. In avirulent BCG infection, the engagement of the P2X7R promotes phagosome-lysosome fusion and induces macrophage apoptosis, thereby causing mycobacterial death The data from two mouse models indicated P2X7R as a key molecule in the development of severe TB and suggested that lung necrosis is enhanced by the activation of this receptor. P2X7R appears to have a dual role in the development of aggressive TB. First, it might facilitate the dissemination of hypervirulent mycobacteria by inducing the lysis of infected macrophages. Second, it might contribute to pneumonia and lung necrosis by promoting widespread cell destruction. Based on these results, we propose that the massive destruction of macrophages by hypervirulent mycobacteria with the help of the P2X7R leads to the release of large amounts of ATP . This re−/− (C57BL/6 background) mice  were bred under specific pathogen-free conditions at the Isogenic Mice Facility . The P2X7R−/− mice were generated by Pfizer Inc. (USA) and backcrossed to C57BL/6 mice for 7 generations; a single nucleotide polymorphism panel analysis throughout the genome suggested a C57BL/6 genetic background (http://jaxmice.jax.org/strain/005576.html). Once infected, the experimental groups were maintained in microisolator cages at the Biosafety Level 3 Mice Facilities  under controlled temperature and humidity and were fed ad libitum.Male 8- to 10-week-old C57BL/6 and P2X7RAll experimental procedures were conducted in accordance with the national regulations on ethical guidelines for mouse experimentation and welfare of the Conselho Nacional de Saúde and Colégio Brasileiro de Experimentação Animal . The protocols were approved by the Animal Care Committee of ICB-USP and UENF under the permit numbers 0026/2009 and 0020/2006, respectively.Mtb strain of the Beijing genotype (Beijing 1471) was isolated from a TB patient at the St. Petersburg Physiopulmonology Research Institute, Russia Mbv isolate (MP287/03 - SB0295 spoligotyping) and the Mtb reference strain (H37Rv - ATCC), respectively. Mycobacteria from a single colony-forming unit (CFU) were suspended in Middlebrook 7H9 medium  that was supplemented with 10% albumin-dextrose-catalase (ADC) (Difco) and 0.05% Tween 80 , with (for Mbv) or without (for Mtb) 0.4% sodium pyruvate (Sigma-Aldrich), and were frozen at −80°C in aliquots of 108 bacilli/ml. Prior to performing the experiments, the aliquots were thawed, diluted in complete 7H9 medium, sonicated and incubated at 37°C for 7 days. To avoid bacterial clumps, the aliquots were sonicated for 1 min, homogenized and kept at rest for 10 min at room temperature (RT). The suspensions were collected, and the absence of bacterial clumps was monitored by microscopic examination. The bacilli were quantified by spectrophotometry at 600 nm.The Mbv strains. CFU numbers were determined after a 3 week incubation at 37°C.The live bacterial burdens were determined by serial dilutions and the plating of cell or tissue homogenates in Middlebrook 7H10 medium (Difco), which was supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC) (Difco); 0.4% sodium pyruvate was added for the Mice were anesthetized by intraperitoneal injections of ketamine  and xylazine , which were diluted in sterile saline. For each mouse, the trachea was exposed via a small midline incision, and 60 µl of the bacterial suspension (approx. 100 bacilli) was inoculated intratracheally (i.t.) The relative mass (organ weight in infected mice/control mice) of each organ was determined. Tissue samples from the right lung, liver and spleen were fixed with 10% buffered formalin, photographed and embedded in paraffin. Serial 4–5-µm sections were stained with hematoxylin-eosin (HE) dye to analyze the tissue alterations and by the Ziehl-Neelsen (ZN) method to detect BAARs. The samples were examined with a Leica microscope (Germany), and images were captured with a Coolpix P995 Nikon camera (Japan).The percentages of the lung tissue occupied by intralveolar space were determined as previously reported for intravascular space in the placenta 4Cl, 0.0169 M TRIS base, pH 7.4) at 37°C in a 5% CO2 atmosphere for 5 min.The left lungs were washed with sterile PBS and placed in Petri dishes with RPMI 1640 medium . The dissected lung tissue was incubated in medium that contained Liberase  and type IV bovine pancreatic DNAse  at 37°C for 45 min. Cells were dispersed with a 10-ml syringe (BD Biosciences) that was fitted with an 18-gauge needle and filtered with a cell strainer . Red blood cells were depleted with a lysis buffer (0.144 M NH6) were stained using appropriate combinations of Pacific Blue-, APC-Cy7-, PE-Cy7-, PE- or V500-labeled monoclonal antibodies (mAbs) to CD11b (ICRF44), CD19 (1D3), CD4 (RM4-5) and CD8 (53-6.7) . Cells were fixed with 2% paraformaldehyde and analyzed by flow cytometry  with FlowJo software.Lung infiltrating cells (1×104 cells/well) were suspended in complete RPMI 1640 medium (Gibco) with 1 mM sodium pyruvate, 2 mM glutamine, 0.05% gentamicin and 10% fetal calf serum (FCS) and were cultured in 96 well-plates (Corning) at 37°C in a 5% CO2 atmosphere for 48 h. Cell culture supernatants were harvested, filter sterilized and stored at −80°C. The levels of IL-1β, IFNγ and IL-10 in the cell culture supernatants were measured with a Fluorokine kit  and a Bioplex system .The lung infiltrating cells  that contained 10 ml of complete DMEM-F12 medium that was supplemented with 20% L929 cell-conditioned medium as a source of macrophage colony-stimulating factor. After 3 days, an additional 10 ml of L929 cell-conditioned medium was added. Four days later, BMDMs were detached from the flasks with 4 ml of 2 mM trypsin in PBS. The viability of BMDMs was higher than 95% over a week period and decreased thereafter.BMDMs were generated from mouse femurs as described previously in vitro experiments were performed in a time window of 6 days after macrophage differentiation. BMDMs (4×104 cells) were cultured in 96-well plates in DMEM-F12 medium with 2% FCS at 37°C in a 5% CO2 atmosphere. To evaluate intracellular bacterial growth, BMDMs were infected at an MOI of 1 for 3 h. After washing 3 times with PBS, the intracellular bacteria were evaluated by performing CFU counts of BMDMs that were lysed with 0.1% saponin (Sigma-Aldrich) for 10 min. To analyze the cytotoxicity induced by the bacteria, BMDMs were infected and cellular viability was determined by acridine orange and ethidium bromide  incorporation using an inverted NIKON TS-100 fluorescence microscope (Japan). To establish the best experimental condition with which to discriminate the differences between experimental groups, the cellular viabilities were systematically determined at MOIs of 10 and 20. The effects of eATP on bacterial intracellular killing and release were assessed as previously described The 4 cells) were infected at an MOI of 10, washed 3 times with PBS and cultured for 4 days in 96-well plates in DMEM-F12 medium with 2% FCS at 37°C in a 5% CO2 atmosphere. Non-infected BMDMs were stimulated with actinomycin D  for 6 h as a positive control for apoptosis and with eATP (5 mM) for 24 h as a positive control for necrosis. BMDMs were stained with FITC-labeled annexin V 2, 1.8 mM CaCl2) for 20 min and then stained with propidium iodide . The samples were analyzed in an inverted NIKON TS-100 fluorescence microscope.BMDMs (4×10p<0.05 (5%). The simultaneous effects of two factors were analyzed by two-way ANOVA and the Bonferroni post-hoc test. One-way ANOVA and the Tukey post-hoc test were used to assess the effects of only one parameter. Survival curves were analyzed with the log-rank test of the Kaplan-Meier method.Statistical analyses were performed with the GraphPad Prism 4 software , and the differences between the groups were considered significant when Figure S1−/− and C57BL/6 BMDMs infected with hypervirulent mycobacteria.Kinetics of intracellular bacillus growth and macrophage necrosis in P2X7R C57BL/6 and P2X7R−/− BMDMs were infected with H37Rv Mtb, Beijing 1471 Mtb and MP287/03 Mbv at an MOI of 1 (intracellular bacillus growth) or at an MOI of 20 (macrophage necrosis). (A) On days 0, 3 and 6 p.i., intracellular bacillus growth was assessed by CFU quantification. (B) Macrophage necrosis was evaluated by AO/EthBr incorporation on days 0, 2, 4 and 6 p.i. at an MOI of 20. Significant differences were observed between the C57BL/6 and P2X7R−/− BMDMs (**p<0.01 and ***p<0.001). The data represent the means ± SD of samples in triplicate. The data are representative of three separate experiments.(TIF)Click here for additional data file.Figure S2−/− and C57BL/6 BMDMs infected with hypervirulent mycobacteria.Annexin V and PI staining in P2X7R C57BL/6 BMDMs were infected with H37Rv Mtb, Beijing 1471 Mtb and MP287/03 Mbv at an MOI of 10. Non-infected BMDMs were stimulated with actinomycin D (2.5 µg/ml) for 6 h as a positive control for apoptosis and with eATP (5 mM) for 24 h as a positive control for necrosis. Photos show BMDM cultures . The data are representative of three separate experiments.(TIF)Click here for additional data file.Figure S3−/− BMDMs.Extracellular ATP-induced necrosis in C57BL/6 and P2X7R C57BL/6 and P2X7R−/− BMDMs were cultured for 24 h with 0, 1, 2, 3 and 5 mM eATP. Macrophage necrosis was determined by AO/EthBr incorporation. Images show viable cells (stained with AO) in green and dead cells with permeabilized membranes (stained with EthBr) in red . The data are representative of three separate experiments.(TIF)Click here for additional data file.


Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining  receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization – homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions.In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining  surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions.These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.The online version of this article (doi:10.1186/s13041-015-0167-1) contains supplementary material, which is available to authorized users. Extensive investigations have characterized mechanisms underlying constitutive and regulated internalization of cell surface proteins, which include pathways dependent or independent of clathrin or caveolae –3. The iThe NMDAR is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions –10. NMDAS)-3,5-Dihydroxyphenylglycine  [Regulated NMDAR internalization causes significant reductions in whole-cell currents mediated by NMDARs –14 and  and e. H, 50 μM) , 14. Con, 50 μM) –17, appl, 50 μM) . We conf, 50 μM) , 18, 19., 50 μM) , 18, 19 2SO4 and Cs2SO4 was used in all of the reported biochemical and electrophysiological experiments. Consistent with previous findings [NMDAR single-channel currents recorded in the cell-attached patch configuration were evoked with NMDA 10 μM) and glycine (3 μM) added into the patch pipette , mean open time (to) and the duration of bursts (Bs), clusters (Cs) and super-clusters (Sc) of recorded NMDAR channels (n = 11 patches) were 0.02 ± 0.007, 1.7 ± 0.2 ms, 2.8 ± 0.4 ms, 7.6 ± 1.3 ms, and 95 ± 33 ms, respectively. After the bath application of DHPG, Po, to, Bs, Cs, and Sc were significantly reduced , DIP co-application significantly reduced the inhibition of NMDAR channels induced by bath application of either DHPG  and a low concentration of glycine (3 μM), which does not cause NMDAR internalization   of NMDAR phosphorylation. We found that after 10 min treatment with high NMDA/glycine there were significant reductions in surface expression of these receptors  into neurons produced a dose-dependent inhibition of NMDAR currents recorded in the whole-cell configuration  were 0.033 ± 0.013, 1.5 ± 0.07 ms, 2.3 ± 0.2 ms, 5.9 ± 1.4 ms, 53.1 ± 21 ms, respectively. Consistent with findings in neurons treated with staurosporine (1 μM) (data not shown), no significant reduction in Po, to, Bs, Cs or Sc was found after bath application of 100 μM glycine in the presence of 1 mM NMDA  in cultured hippocampal neurons without or with knockdown of PKD1 were recorded Fig.  and f. IIn order to determine whether the functional properties of remaining surface NMDARs, which are not internalized, may be altered by internalization of remote NMDARs, we conducted single channel recordings in the cell-attached configuration. Previous studies have demonstrated that NMDAR activity can be regulated by remote NMDARs through intracellular signaling pathways , 21, 59.It was thought until now that through reducing the number of active receptor/channels located on the cell surface, the regulated receptor/channel internalization reduced the receptor/channel-mediated responses. Our single channel data showed that the NMDAR internalization, which reduced the amount of NMDARs expressed on the neuronal surface, also led to inhibition of the activity of remaining surface NMDARs. Furthermore, the inhibition of activity was mediated through intracellular PKD1 signaling as we found that knockdown of PKD1 prevented the inhibition of single channel activity of remaining NMDARs induced by NMDAR internalization, and also prevented the reduction of synaptic NMDAR responses Fig. . Thus, oA large amount of data have demonstrated that the activation of group 1 metabotropic glutamate receptors through application of DHPG is an important mechanism underlying the induction of long-term depression of AMPAR-mediated synaptic responses (LTD) –17, 60. NMDARs are well-known to undergo two types of regulated internalization  – homoloThe phenomenon discovered in our work underlying the regulation of remaining surface receptor/channels by the endocytosis of the same type receptor/channels might therefore be NMDAR-specific. Detailed mechanisms underlying how PKD1 signaling is activated by NMDAR endocytosis and what role, if any, is played by other receptors such as AMPAR in the PKD1 activation remains to be clarified. Further clarifying the mechanisms underlying the regulation of remaining surface receptor/channels is challenging and required for understanding of the principal biological event: internalization of cell-surface receptor/channels. Altogether, we conclude that the regulated NMDAR internalization causes down-regulations of the activity of remaining  surface NMDARs and the synaptic transmission through serine phosphorylation of remaining surface NMDARs induced by PKD1 activation Fig. .Previous studies, including ours, have shown that tyrosine phosphorylation may enhance NMDAR activity while tyrosine dephosphorylation may down-regulate NMDAR activity –66 and tC-termini of NMDAR subunits are known to be critically involved in regulation of both the NMDAR internalization and gating , 73, 74.Caenorhabditis elegans [Taking together with findings that the application of active recombinant PKD1 protein into cells produced inhibition of NMDAR currents and that this inhibition was blocked by S1416F mutation in the GluN2A subunit or C-tail truncation in the GluN2B subunit, we conclude that regulated NMDAR internalization acts as an upstream signal which activates the downstream PKD1 and thereby causes the phosphorylation and down-regulation of remaining surface NMDARs Fig. . PKD1 is elegans .The NMDAR internalization-induced inhibitions of both the remaining surface NMDAR activity and the NMDAR-mediated synaptic response can be significantly reduced by knockdown of PKD1. This finding has not only revealed a role played by remaining surface NMDARs in the regulation of NMDAR functions by NMDAR internalization, but also provided a novel mechanism that quantity changes in cell surface receptors may down-regulated the receptor-mediated whole-cell response through depressing the activity of non-internalized surface receptors.Thus, we conclude that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs expressed on the cell surface but also causes an inhibition of the activity of non-internalized NMDARs through the activation of PKD1 Fig. . The funAll animal experiments were conducted following the guidelines of the Canadian Council on Animal Care and the NIH, and approved by the Animal Care Committee at University of Toronto and Animal Care and Use Committee at Florida State University.4 cells/cm2) or HEK293 cells (6 × 104 cells/cm2) was plated onto 60 mm culture dishes coated with poly-D-lysine. Neurons were cultured with Neurobasal™ media  supplemented with B27 (2 %), bFGF2 (2 ng/mL) and L-glutamine (0.5 mM), and HEK293 cells with Dulbecco’s Modified Eagles Media (DMEM)  supplemented with 10 % fetal bovine serum. For imaging experiments, a low density of cells (approximately 3 × 103 cells/cm2) was cultured on glass cover-slips . For electrophysiological recordings in neurons a low density culture of hippocampal neurons was plated in poly-D-lysine-coated dishes with Neurobasal™ media as mentioned above, or on collagen-coated dishes with minimum essential media (MEM) supplemented with 10 % horse serum. Since no difference was found in electrophysiological studies of neurons cultured with MEM versus those with Neurobasal™ media, the data were pooled. For electrophysiological recordings in HEK293 cells, low density culture was maintained in DMEM supplemented with 10 % fetal bovine serum.Methods for primary neuron culture and HEK293 cell culture have been described previously , 83, 84.Methods for constructing cDNAs of NMDAR subunits were described in our previous reports , 83. Theg for 5 min at 4 °C. Then, the supernatant (300 μl) was incubated with an NMDAR subunit antibody or with NeutrAvidin agarose  to precipitate NMDAR subunit protein or biotinylated protein. NMDAR proteins expressed on cell surface were determined through detecting biotinylated NMDAR subunit protein in NMDAR immunoprecipitates or NMDAR proteins in pull downed proteins with NeutrAvidin agarose.Surface protein internalization induced by bath treatment with NMDA (1 mM) and glycine (100 μM) (defined as high NMDA/glycine in this article) for 10 min or with DHPG (50 μM) for 5 min were determined with biochemical assays and imaging observations. For biochemical assays, cell surface proteins were biotinylated with cell impermeant non-cleavable EZ-Link Sulfo-NHS-LC-LC-Biotin , as described previously , 86. In For image analysis, neurons cultured on glass cover-slips were biotinylated as mentioned above. Following treatment with the vehicle, high NMDA/glycine (for 10 min) or DHPG (for 5 min) at 37 °C, neurons were incubated with biotin for 1 h at 4 °C. And then neurons were fixed with ice-cold 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100 and incubated with an antibody against the GluN1 , GluN2A , or GluN2B  subunit. Biotinylated neuronal surface proteins were visualized with streptavidin-conjugated Alexa 488 (green), and NMDAR subunit proteins were visualized with Alexa 568-conjugated goat anti-mouse or Donkey anti-rabbit IgG. Background staining was removed by thorough washing with PBS. Coverslips were then mounted using Fluoromount® (Sigma). Z-stack images of fluorescence staining were taken under an Axiovert 200 M fluorescence microscope (Carl Zeiss) equipped with CCD camera  using software AxioVision 4.5 (Carl Zeiss). Co-localization of staining of the GluN1, GluN2A or GluN2B subunit with biotinylated cell surface proteins was analyzed using NIH-ImageJ software  . Ratios g for 15 min at 4 °C to remove the nuclear pellet and other large debris (P1). The supernatant (S1) was collected for further experiments to yield the crude synaptosomal fraction (P2) which was subsequently lysed by hypoosmotic shock in 0.5 ml of ice cold 4 mM HEPES (pH 7.4) plus protease/phosphatase inhibitors and three strokes of an eppendorf tube homogenizer. It was kept on ice for 30 min to ensure complete lysis. By using a pasteur pipette, the lysate was layered on top of a discontinuous gradient containing 0.8 to 1.0 to 1.2 M sucrose (top to bottom) in a clear tube, and was centrifuged at 150,000x g for 2 h at 4 °C. LP1 was recovered in the layer between 1.0 and 1.2 M sucrose, and then it was diluted to 0.32 M sucrose by adding 2.5 volume of 4 mM HEPES pH 7.4. The synaptic plasma membrane was pelleted by centrifugation at 150,000 g for 45 min at 4 °C and then resuspended in cell lysis buffer (mM): Tris–HCl , NaCl (150), 1 % Igepal CA-630, 0.5 % deoxycholate, 0.1 % SDS, EDTA (2), NaF (50), sodium pyrophosphate (10), sodium glycerophosphate (20), sodium orthovandate (4); pepstatin A (20 μg/ml), leupeptin (20 μg/ml), aprotin (20 μg/ml), PMSF (1). The protein concentration was determined by Bradford assay. NMDAR subunit proteins were immunopurified from the LP1 fraction and their serine phosphorylation was examined by anti-pSer antibody (Invitrogen) in Western blot analysis.To determine the effect of NMDAR endocytosis on phosphorylation of surface NMDAR proteins, the synaptic plasma membrane (LP1) , 30 was g for 15 min. 20 μl of supernatant was used to measure total GluN1, GluN2A or GluN2B. 260 μl of supernatant was incubated with 100 μl of 50 % NeutrAvidin agarose (Thermo Scientific) at 4 °C for 3 h. The agarose beads were washed three times with cell lysis buffer and the bound biotinylated proteins were eluted from the beads in 40 μl of 2 × Laemmli sample buffer by boiling.Immunoprecipitation and Western blotting were performed as described previously , 83. SolThe majority of the gels used for the Western blot analysis were 7.5 % polyacrylamide SDS-PAGE gels with Tris-glycine running buffer. Synthetic peptides of S1407-H1419 or S1407-H1419 (pS1416) were loaded on precast 4–12 % Bis–Tris polyacrylamide gels with MES running buffer  for the analysis of the specificity of the anti-pS1416 antibody. The filters were stripped repeatedly and probed successively with various antibodies. Antibody staining bands were visualized with Enhanced Chemiluminescence  or Immobilon (EMD Millipore). The band intensities were quantified with NIH-imageJ software . Antibodies used in this study include anti-GluN1 , anti-GluN2A C-terminal , anti-GluN2A N-terminal , anti- GluN2B , anti-pSer , anti-pS1416 , anti-Kv3.1 , anti-actin , and antibodies recognizing the phosphorylated S916 in PKD1   biochemical experiment. We conducted careful, unbiased, densitometry analysis of all western blots by using NIH-ImageJ software. The relative densitometric intensities on the Western blots of the treated samples compared to samples treated only with culture medium were calculated, and multiplied by 100, as “Percent of Control”. These values were used to show the effects of any treatment. Ratios of the protein band intensities of the biotinylated GluN1, GluN2A or GluN2B subunit detected with streptavidin versus that with the antibodies were calculated to determine the amount of surface NMDARs and normalized to the ratio detected in cells treated only with culture medium conducted in parallel. The immunoprecipitation experiments from non-biotinylated tissues were conducted as negative controls.6 transducing units per ml (Open Biosystems/Thermo Fisher Scientific). The viral stock was aliquoted and stored at −80 °C. Nine to ten days after plating, cultured neurons were infected by adding the viral-supplemented medium (stock) into culture medium . Four to five hours after infection, neurons were washed with 1X PBS and cultured for 7–10 days before electrophysiological or biochemical experiments. According to image observation more than 80 % of cells were infected.Lentiviral particle production and infection were performed as recommended by the manufacture . For Lentiviral particle production, HEK293T cells were co-transfected with the Trans-Lentiviral packaging mix and the transfer vector containing green fluorescent protein (GFP) cDNA and the PKD1 shRNA (TGCTGTTGACAGTGAGCGCATCGTTCACTGTGACCTCAAATAGTGAAGCCACAGATGTATTTGAGGTCACAGTGAACGATATGCCTACTGCCTCGGA) or control shRNA (TGCTGTTGACAGTGAGCGATCTCGCTTGGGCGAGAGTAAGTAGTGAAGCCACAGATGTACTTACTCTCGCCCAAGCGAGAGTGCCTACTGCCTCGGA) (Open Biosystems/Thermo Fisher Scientific) as recommended by the manufacture (Open Biosystems/Thermo Fisher Scientific). The virus-containing supernatants were collected 48–72 h post-transfection, centrifuged and filtered through 0.45 μm low protein binding filter with the approximate titer 1 × 102SO4 (100), Cs2SO4 (10), CaCl2 (4.8), HEPES (25), glucose (32), TTX (0.001), glycine (0.003), bicuculline (0.01) and strychnine (0.01), pH: 7.35 and osmolarity: 310–320 mOsm. Free Ca2+ concentration in this solution was at 1.2 mM as confirmed by measurement with a Ca2+ selective electrode . To prevent cell damage during NMDAR activation, in this solution, Cs+ and SO42− replaced K+ and Cl−, respectively [Methods for recordings of NMDA-evoked single-channel or whole-cell currents, and mEPSCs have been described previously , 83, 84.ectively , 89. Recc) where each tc was defined by 1-e-tc/τs = e-tc/τm (τs and τm: the time constants of short and intermediate gap lengths) [Single-channel currents were recorded with a patch potential of 70 mV from the reversal potential using an AxoPatch 200B amplifier. Recorded electronic signals were filtered at 10 KHz  and sampled continuously onto a computer at 20 KHz with the pClamp9 software . Consistent with previous observations , 90, moslengths) , 90.To examine effects of DHPG application, after a 3 to 5 min period of control recordings, DHPG (50 μM) was bath applied for 5 min and then recordings were continued for another 10 to 20 min. To examine effects of high NMDA/glycine application, the extracellular solution containing 1 mM NMDA was applied after the cell-attached configuration was formed. Control recordings were conducted for 3–5 min in neurons bathed with the extracellular solution containing 1 mM NMDA, and then glycine (100 μM) was bath co-applied.2 (2), K-ATP (4), osmolarity 290–300 mOsm (DC resistance: 4–7 MΩ). NMDA-mediated whole-cell currents were evoked by application of 1 mM NMDA dissolved in the extracellular solution via a computer-controlled multi-barrel fast-step perfusion system . Whole-cell currents were recorded using Axopatch 200B amplifiers  under the voltage-clamp condition at a holding potential of −60 mV except where indicated. On-line data acquisition and off-line analysis were performed using pClamp9 software (Molecular Devices). To determine effects produced by intracellular delivery of PKD1 added in the intracellular solution filling recording electrodes, recorded whole-cell currents evoked by NMDA application were compared with that recorded immediately after breakthrough (defined as “0” min in this work).For whole-cell voltage-clamp recordings cells were bathed with the same standard extracellular solution mentioned above except that 10 μM glycine was applied for recordings in HEK cells. Recording electrodes were filled with intracellular solution composed of (in mM): CsCl (145), BAPTA (0.5), HEPES (10), MgClMiniature excitatory post-synaptic currents (mEPSCs) were recorded in cultured hippocampal neurons  except for those as indicated.Multiple tests for examining normality or variance of data were performed to determine which type of statistic tests could be used to examine differences between experimental groups. For comparisons of channel activity before and/or after bath application of any agents, paired two-tailed Student’s t-tests were performed. For comparisons of protein expression and phosphorylation with that in the group of cells treated only with culture medium, independent two-tailed Student’s t-tests were performed. Repeated measures analysis of variance (ANOVA) were used for comparison of experiments involving time courses. All data are expressed as mean ± SEM.


Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve  Structural factors regulating HSC proliferation remain poorly investigated . Inside ractions .in vitro in the presence of thrombopoietin (TPO), an essential cytokine for HSC proliferation and differentiation, acting as a MK proliferation, maturation and differentiation agonist [In the megakaryocytic lineage, megakaryocyte (MK) progenitors proliferate and differentiate into MK precursors that become polyploid by endomitosis, i.e. DNA replication without cell division . Platele agonist .in vitro remains a challenging task, requiring synergy between biochemical and biophysical factors to reach high platelet yields. Thus, in the aim to improve in vitro platelet production, several novel approaches are currently developed. Hemodynamic shear forces contribute to proplatelet and platelet formation from mature MK in vivo [in vitro [in vitro is accelerated and concentrated in flow conditions [ex vivo in liquid culture, except for the regulatory activities normally provided by the structural bone marrow microenvironment [ex vivo from HSC using 3D scaffolds of nonwoven polyester fabric and inverted colloidal crystals/polyacrylamide porous hydrogels, but these platelets seemed activated [In the bone marrow, interactions between bone cells, hematopoietic cells and vascular cells regulate hematopoiesis. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis, the final step of MK differentiation . This pr in vivo  and in vin vitro . We reponditions , a procenditions ,11. A synditions , ii) MK nditions , and iiinditions . Indeed,ironment . Even ifironment ,15,16, iironment ,18. So f+ cell proliferation but also on MK differentiation. We found that on one hand, 3D environment delayed MK progenitors (CD34+/CD41-) differentiation into MK precursors (CD34-/CD41+) leading to mature MK (CD41+/CD42b+). On the other hand, 3D environment improved enlargement of these mature MK allowing increased platelet production in flow conditions.In this study, we examined how a porous 3D structure affects HSC proliferation, MK differentiation and platelet production. The results clearly demonstrate the effect of 3D environment not only on CD34+ cell culture in comparison to liquid cell culture.3D scaffolds were prepared from polysaccharide-based hydrogels as previously described ,20. HydrBacillus acidopullulyticus, 44.4 U/mL; Sigma-Aldrich, Saint-Quentin Fallavier, France) and dextranase  were added to 3D hydrogels for 30 minutes at 37°C, unless otherwise stated. Following scaffold degradation, cells were recovered by centrifugation for 10 minutes at 290g and compared to cells cultured in liquid conditions (liquid-culture cells). Controls consisted of liquid-culture cells that were added to empty 3D hydrogels immediately before enzymatic treatment. In some experiments, enzymatic treatment of 3D hydrogels was followed by a bovine serum albumin  density gradient centrifugation to separate matrix debris from viable cells . Pullulanase , peripheral blood or bone marrow by using an immunomagnetic technique  [+ cell samples were separated in two aliquots of 300 000 cells, one for liquid culture and one for 3D hydrogel culture. For liquid culture, 150 000 cells/cm2  were added and for 3D hydrogel culture, 3 dry disks were seeded with 100 000 cells each  supplemented with 15% BIT 9500 serum substitute , α-monothioglycerol (Sigma-Aldrich) and liposomes  [CD34 France) . This stAldrich) . Human r-Flow cytometry used for study of differentiation markers and ploidy-Confocal immunofluorescence microscopy-Clonogenic assay-RNA extraction and real-time qPCR-Platelet production in flow assay-Platelet activation and spreading-Statistical data analysisOnline supplemental data available on the PLOS One website described in detail the materials and methods listed below:+ cell proliferation and differentiation. Rehydrated transparent 3D hydrogels were organized into a complex network of large (200 μm) pores (+ cells loaded on top of 3D entered pores within one hour after seeding (not shown). Pore occupancy by proliferating cells was dependent on the initial number of cells, and a condition of 100 000 cells/disk was selected for further experiments  whereas in liquid culture, total cell number increased to reach a peak value of 2.6 x 107 ± 2.75 x 107 on day 9, then decreased until day 16 where total cell number did not differ between the two conditions . After day 16, whereas no cell survived in liquid culture, cells were maintained in 3D up to day 36. Thus, 36 days after initial seeding, large numbers of cells were still present in 3D  pores . UCB-dereriments . Cells meriments , whereaseriments . This wa culture . In orde culture . Betweennt in 3D . Therefo+ cell (MK precursors) number in both conditions. Results showed a peak of total CD41+ cell number on day 12 in liquid culture (2.07 x 107 ± 1.2 x 107) compared with a peak on day 16 in 3D (1.39 x 107 ± 1.2 x 107) followed by MK precursor presence until day 36  but absent in liquid culture. Moreover, CD34+ cells remained in 3D (31.5% ± 9.7% on day 9 and 6.4% ± 4.9% on day 16) while they disappeared in liquid culture   . Interesersisted , while Cersisted . After d+ and CD11b+/CD14+ cells) and macrophages (CD14+ cells) on days 23 and 36  than in liquid culture . Thus, MK progenitors were able to differentiate into more mature larger MK.CFU-MK potential was assessed in clonogenic assays performed with 3D cells recovered on days 16 and 23 and with liquid-culture cells recovered on day 16 . The numTo support our previous results, we analyzed several transcription factors involved in megakaryopoiesis –26. Ther+ cells were stained within the hydrogels. As shown in +/CD42b- cells) and mature MK (CD41+/CD42b+ cells) were both present in 3D  showed a higher ploidy in 3D than in liquid culture , whereas 2N ploidy was significantly increased in liquid culture than in 3D . These results showed that the 3D environment enhanced megakaryocytic maturation by favoring endomitosis.Ploidy of UCB mature MK was further investigated 11 days after seeding by flow cytometry assessment of nuclear markers. Cells were recovered from hydrogels by enzymatic degradation of the pullulan-dextran structure in conditions providing the highest recovery while maintaining cell integrity, as determined by microscopy and analysis of surface markers (data not shown). Ploidy levels determined after selection of mature MK (CD41 culture . Cumulat+ cells in 3D environment (+/CD41- cells (non-megakaryocytic cells and cell progenitors) derived from bone marrow cells  than in liquid culture (6 platelets/CD34+) on day 16. Furthermore, platelet production from 3D mature MK was still observed up to day 36 (data not shown). These results demonstrated that this 3D environment allowed the recovery of many platelets.In order to demonstrate that culture in 3D yielded platelets, platelet production was studied in flow conditions after recovery of mature and viable MK from 3D on day 16. When exposed to a high shear rate in a microfluidic platform , mature in vitro platelet production, several attempts are currently developed [HSC isolated from hematopoietic tissues differentiate into MK progenitors and then into mature MK, whose function is to terminally release platelets that are essential for bleeding arrest. To improve eveloped ,11,16. I+ progenitor cell maintenance and expansion were improved by using 3D scaffolds with pore sizes close to 200 μm [+ progenitor cell proliferation and MK differentiation. TPO is important not only for HSC and hematopoietic progenitor cell maintenance but also for MK differentiation . In 3D Morrison . Indeed,Morrison . This co culture ,25, persntiation ,35,36. P+ cell commitment in other lineages was observed in 3D, as shown by changes of myelomonocytic CD11b and CD14 markers [+ cell commitment in all hematopoietic lineages [+ cells observed in 3D environment is essential to obtain and preserve mature MK but also other mature cells (red blood cells or white blood cells).In addition to the specific effect of 3D environment on MK differentiation, our data show that 3D structures support not only MK differentiation but also erythropoietic and myelomonocytic differentiation. Indeed, CD34 markers ,38 and e markers ; the lat markers . In addilineages . Thus, tvia increased cytoplasm volume [in vitro without studying the influence of 3D environment on platelet formation [in vitro microfluidic platform. Thus, this study demonstrates for the first time the effects of 3D environment on MK differentiation and in particular on the improvement of enlargement of mature MK leading to increase the production of platelets. Furthermore, 3D environment is mainly good to keep a continuous supply of CD34+ cells to undergo MK differentiation.The role of polyploidy for increasing platelet formation has been suggested but it still remains unclear . A gene m volume . Howeverm volume ,43. Highm volume  in maturm volume ,45. In am volume . This F-m volume . Furtherm volume –50. Thusm volume ,11,13,16ormation . In contCollectively, 3D environment promotes cell clustering, sustains and improves MK differentiation into fully mature MK leading to increase the production of functional platelets. This study provides the first direct evidence that spatial organization and biological cues synergize for efficient MK and platelet production. This 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment. Recent studies showed the feasibility of expanding human induced pluripotent stem cells (iPSCs) into MK underline potential therapeutic applications for generation of platelets –53. ThusS1 Table(DOCX)Click here for additional data file.S1 Fig+ cells were seeded inside 3D pores at day 0 in presence of 50 nM TPO and 20 ng/mL SCF. Steps 2 and 3: On days 7, 9 and 12, the disks were transferred in new wells and fresh medium containing 20 nM TPO was added. Step 4: Cells excluded from 3D and accumulated at the bottom of the wells were discarded. Step 5: On days 16, 23, 30 and 36, mature MK were extracted from 3D by enzymatic lysis. Step 6: Platelets were produced by mature MK perfusion in microchannels coated with VWF. Steps 7 and 8: At the end of the perfusion, platelets were collected and platelet functions were assessed. Abbreviations: TPO, thrombopoietin; SCF, stem cell factor; MK, megakaryocytes; VWF, von Willebrand factor.Step 1: CD34(DOCX)Click here for additional data file.S2 Fig(A) Frequency of CD41-/CD11b+/CD14-, CD41-/CD11b+/CD14+ (both myelomonocytic cells) and CD41-/CD11b-/CD14+ (macrophages) cells in 3D  and liquid culture  between day 7 and day 36. (B) Frequency of CD41-/GpA- and CD41-/GpA+ (erythrocytic cells) cells in 3D  and liquid culture  between day 7 and day 36. Data are means ± SEM of 4 independent experiments. *p<0.05. In 3D, late time points (days 23 and 36) were compared to day 7. Results indicate that the effect of 3D environment also results in commitment into the erythropoietic and myelomonocytic lineages during a second wave of differentiation that takes place between day 16 and day 36.(DOCX)Click here for additional data file.S3 Fig(A) Bone marrow and peripheral blood cell proliferation inside 3D pores, 12 days after seeding. Images were acquired using the Axiovert 135 transmission optical microscope with 20X Plasdic magnification. Bar = 20 μm. (B) CD41/CD34 dot plots of one representative experiment in 3D and liquid culture on day 12. Similar results are obtained with neonatal or adult CD34+ cells with the persistence of non-megakaryocytic cells and cell progenitors (CD34+/CD41- cells); these cells could still commit in the megakaryocytic lineage. (C) Ploidy analysis of CD41+/CD42b+ peripheral blood cells in 3D (black bars) compared to liquid culture (white bars). Data are means ± SEM of 3 independent experiments. Results show higher percentages of 8N ploidy classes in 3D cells (25.3% ± 6.1%) than in liquid-culture cells (15.9% ± 4.9%), whereas 2N ploidy was more frequent in liquid culture (48.1% ± 6.3%) than in 3D (39.3% ± 6.4%). Abbreviation: UCB, umbilical cord blood.(DOCX)Click here for additional data file.S4 Fig(A) Representative images of CD41 (green)/F-actin (red) staining on platelets collected after perfusion of 3D mature MK in microfluidic platform. Filopodia (arrowhead) and stress fibers (arrow) are visible on activated platelets. (B) Representative images of PAC1 staining of integrin αIIbβ3 activated (green) and of F-actin staining (red) on platelets collected after perfusion of 3D mature MK in microfluidic platform. Lamellipodia (asterisk) are visible on activated platelets. Images were acquired using the Axio Observer D1 fluorescence optical microscope with 63X Plasdic magnification. Bar = 2 μm.(DOCX)Click here for additional data file.S1 Video-1 for 45 min on VWF-coated microchannels. Different steps of platelet production were visualized in real-time using the Axiovert 135 transmission optical microscope with 20X Plasdic magnification. Digital images were recorded at 0.25 images/s using Replay software (Microvision Instruments). For visualization, Archimed software (Microvision Instruments) was used to grab frames and record at a velocity of 10 images/sec (40-fold acceleration). Bar = 20 μm.The video shows extremely long MK elongations similar to beads-on-a-thread. Proplatelets and platelets are also visible in the last section of the video. Mature and viable MK were recovered from 3D or liquid culture on day 16 and perfused at a shear rate of 1800 s(DOCX)Click here for additional data file.S2 Video-1 for 45 min on VWF-coated microchannels. Different steps of platelet production were visualized in real-time using the Axiovert 135 transmission optical microscope with 20X Plasdic magnification. Digital images were recorded at 0.25 images/s using Replay software (Microvision Instruments). For visualization, Archimed software (Microvision Instruments) was used to grab frames and record at a velocity of 10 images/sec (40-fold acceleration). Bar = 20 μm.Compared to mature MK obtained from 3D culture, notice the shorter elongations interacting with the surface during the first part of the video and the reduced production of proplatelets and platelets during the second part of the video. Mature and viable MK were recovered from 3D or liquid culture on day 16 and perfused at a shear rate of 1800 s(DOCX)Click here for additional data file.S1 Materials and Methods(DOCX)Click here for additional data file.


AG) repeats. Moreover, mutant huntingtin protein translated from expanded r(CAG) also yields toxic effects. To discern the role of mutant RNA in these diseases, it is essential to gather information about its structure. Detailed insight into the different structures and conformations adopted by these mutant transcripts is vital for developing therapeutics targeting them. Here, we report the crystal structure of an RNA model with a r(CAG) motif, which is complemented by an NMR-based solution structure obtained from restrained Molecular Dynamics (rMD) simulation studies. Crystal structure data of the RNA model resolved at 2.3 Å reveals non-canonical pairing of adenine in 5´-CAG/3´-GAC motif samples in different syn and anti conformations. The overall RNA structure has helical parameters intermediate to the A- and B-forms of nucleic acids due to the global widening of major grooves and base-pair preferences near internal AA loops. The comprehension of structural behaviour by studying the spectral features and the dynamics also supports the flexible nature of the r(CAG) motif.In humans, neurodegenerative disorders such as Huntington’s disease (HD) and many spinocerebellar ataxias (SCAs) have been found to be associated with CAG trinucleotide repeat expansion. An important RNA-mediated mechanism that causes these diseases involves the binding of the splicing regulator protein MBNL1 (Muscleblind-like 1 protein) to expanded r(C RNA plays an essential role in normal and abnormal cell functions, which makes it a crucial target for the selective binding of small molecules . Due to AG) repeats that corrects splicing defects in disorders caused by RNA gain-of-function [In addition to the CAG repeats located in coding regions, expansion in UTRs also causes pathogenesis –16. Suchfunction . These sfunction ,26. Thes2. This A-A interaction causes local unwinding of RNA duplexes in which adenosines adopt an anti conformation with a single H-bond between the C2H2 and N1 of paired adenosines [anti conformation and the N6 amino group in an H-bond with N1 [Over the past decade, the structural characteristics of RNAs containing CAG repeat motifs were explored by biochemical methods –31. Thesenosines . Similar with N1 .AG)3GUCC)2, with these loops favouring the anti-anti conformation. However, simulation studies have shown that these 1x1 AA nucleotide internal loops are stable in the syn-anti conformation. Furthermore, this conformation is affected by the binding of ions or small molecules to 1x1 AA nucleotide internal loops [However, neither of these studies reveal the dynamic behaviour of the adenosines, which is likely to be observed in motions involving large amplitude changes. In contrast, another group reported the crystal structure of self-complimentary duplex RNA containing CAG repeats resolved at 1.65 Å resolution. This study revealed the dynamic behaviour of AA internal loops in 5´ r simulation studies.AGCAGCAGGUCC)2 was purchased from Integrated DNA Technologies, Inc. in a desalted and deprotected form. Purification of the RNA was performed using a Waters HPLC instrument with an attached UV-Vis detector. The RNA was suspended in water and passed through an XTerra Prep MS C18 HPLC column . Elution was performed by applying a linear gradient (from 100 to 0% 10 mM triethylammonium acetate (pH = 7) in acetonitrile over 55 minutes) with a flow rate of 2 ml/min (tR = 25 mins). Subsequent desalting was performed by a Sephadex PD-10 prepacked size-exclusion column. The final concentration of the RNA duplex was calculated from its absorbance at 260 nm (at 95°C). Hyther Server, based on nearest neighbour thermodynamics, was used to determine the molar extinction coefficients [The CAG motif containing RNA 5´ r, 25 mM magnesium sulphate and 1.8 M ammonium sulfate using the sitting drop vapour diffusion method within 2–3 days at 18°C.A complete diffraction dataset at 2.3 Å resolution was collected using liquid nitrogen-immersed flash-frozen crystals. ADSC CCD detectors on beamline 9–1 at SSRL or beamline LS-CAT (21-ID) at the Advanced Photo Source  under cryoconditions (100 K) were used to collect the data. The data were scaled and integrated using HKL2000 . InitialElectrostatic potential was calculated from the corresponding crystal structure model. The non-linear Poisson Boltzmann equation  was used3DNA, a software package, was used to calculate helical parameters, torsional angles and groove widths . Vector-AGCGG)2, and three CAG motifs, 5´ r(GGGCCAGCAGCAGGUCC)2, were used for NMR spectroscopy. RNA samples were prepared by resuspending lyophilised RNA in 10 mM phosphate buffer, pH 7.2, 0.1 M NaCl, and 50 mM EDTA in 10% D2O. NMR spectra were recorded with a Bruker NMR spectrometer with field strengths of 400 and 500 MHz. One dimensional proton spectra were recorded at various temperatures. Spectral assignments were performed using NOESY (Nuclear Overhauser Effect SpectroscopY), DQF-COSY (Double Quantum Filtered Correlation SpectroscopY) and TOCSY .RNA duplexes with a single CAG motif, 5´ r NOESY was performed acquiring 4096 data points with 64 transients for each of the 296 FID signals. The spectra were recorded at 288, 298 and 308 K with mixing times of 300, 200 and 100 ms. A NOESY spectrum was recorded using an excitation exculpating pulse sequence to suppress residual water signals.1H-1H NOE distances. These distances were used to restrain the RNA duplex for restrained Molecular Dynamic simulation studies.SPARKY was used to visualise the spectra and calculate The minimisation and restrained Molecular Dynamics (rMD) simulation were performed using Discovery Studio 3.5 . The RNA duplex was built using the Macromolecule module in Discovery Studio 3.5. After typing the duplex with a CHARMM force field , the modAfter minimisation and optimisation, the RNA duplex was subjected to Standard Dynamic Cascade. Within this cascade, the system was again minimised with a steepest descent minimisation of 500 steps followed by a conjugate gradient minimisation of 500 steps. The system was then subjected to gradual heating from 50 to 300 K for 4 picoseconds. The molecule was equilibrated for 10 picoseconds with a time step of 2 femtoseconds at a constant temperature of 300 K. Equilibration was followed by production runs under a constant temperature (300 K) with PME treatment for electrostatic interactions, SHAKE for bonds containing hydrogen and a 2 femtosecond time step. Simulations were performed for 25 nanoseconds, and 100 conformations were generated. The output files were analysed with Discovery Studio 3.5 based on properties such as potential energy of the system, root mean square deviation (RMSD) and the presence of hydrogen bonds.Here, we report the crystal structure of RNA duplexes containing three consecutive 5´-CAG/3´-GAC motifs showing dynamics, which is further validated by NMR studies. The duplex crystallised in the double-stranded helical structure and the structure was refined to resolution of 2.3 Å.anti conformation. Despite being in the anti conformation, one of the A's in the central AA internal loop was slightly tilted and did not have well-defined electron density  and a B-form DNA duplex, the inclination angles of the bases were found to be lower compared with the A-form of RNA. Such changes in the inclination of bases could be derived from stacking purine interactions . Thus, aAG) repeat RNAs. The electrostatic distribution shows that these repeats have a larger density of partial positive charges in the minor groove 2 at various temperatures. With an increase in temperature, the broadening of proton resonance peaks in the imino region (G6NH) was observed 2 duplex in NOESY spectra collected at 300 ms mixing time at two different temperatures, 288 and 308 K, show that the interproton distance between A5H8 and G6H8 increases with increasing temperature. This result indicates the existence of adenine conformational dynamics. In addition, the A5H1´- A5H2 cross peak intensity also varied with a change in temperature 2, based on distances obtained from NOESY experiments. The duplex was restrained based on the 1H-1H NOE distances and Watson-Crick base pairing. The duplex was minimised to the lowest energy conformation and simulated using restrained Molecular Dynamics. One hundred distinct structures were generated in a 25 nanosecond simulation run.To explicitly examine the conformational flexibility and dynamic nature of AA pairs, rMD simulations were performed using a RNA construct containing a single CAG motif, 5´ r3GUCC)2 showing imino proton region at variable temperature. Asterisk mark attributes to the appearance of new resonance due to the dynamics in adenine pairs.One dimensional proton spectra for 5´ r(UUGGGC(C(TIF)Click here for additional data file.S2 FigAG)3GUCC)2 showing base and sugar 1´ region at variable temperature.One dimensional proton spectra for 5´ r(UUGGGC(C(TIF)Click here for additional data file.S3 FigNOESY spectra showing NH-NH NOEs at 298K. Some of the peaks are overlapped.(TIF)Click here for additional data file.S4 FigNOESY spectra showing base to sugar 1´ correlation at 298K showing. Some of the peaks are overlapped.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.S14 Table(DOCX)Click here for additional data file.


This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival.In analogy with sepsis, current postcardiac arrest (post-CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVOA prospective, observational study during therapeutic hypothermia (24 hours -33°C) in 82 post-CA patients monitored with near-infrared spectroscopy.P = 0.04). The mean SVO2 range associated with maximal survival was 67 to 72% , P = 0.001). In two separate multivariate models, a mean MAP , P = 0.02) and a mean SVO2 , P = 0.01) in the optimal range persisted as independently associated with increased survival after correction for presence of early bystander CPR and presenting shockable rhythm. Based on more than 1,625,000 data points, we found a strong linear relation between SVO2 (range 40 to 90%) and average cerebral saturation (R2 = 0.86) and between MAP and average cerebral saturation for MAPs between 40 and 87 mmHg (R2 = 0.70). Based on our hemodynamic model, the optimal MAP and SVO2 were determined to be 87 mmHg and 72%.Forty-three patients (52%) survived in CPC 1 to 2 until 180 days post CA. The mean MAP range associated with maximal survival was 76 to 86 mmHg , 2 (72%) and MAP (87 mmHg) derived from our hemodynamic model matched with the observed SVO2 (67 to 72%) and MAP (76 to 86 mmHg) associated with maximal survival. Prospective interventional studies to reach or maintain these targets are needed to confirm these findings.The optimal SVO


Rb is a tumor suppressor, and regulates various biological progresses, such as cell proliferation, development, metabolism and cell death. In the current study, we show that Rb knockout in 3T3 cells leads to oxidative redox state and low mitochondrial membrane potential by regulating mitochondrial activity. Our results indicate that Rb plays an important role in controlling redox homeostasis. More importantly, the functions of Rb in modulating cell proliferation, death and transformation are, at least in part, mediated by its controlling cellular redox state. In addition, our results also suggest that the cellular redox state possibly determines various biological activities, including cell survival, death and transformation, where Rb is functioning as a regulator of redox homeostasis. The retinoblastoma protein (Rb) is a well known tumor suppressor, and functions in the control of cell cycle progression and proliferation Besides the canonical pathways that link Rb tumor suppressor to human cancers, recent studies have shown an essential role for Rb in the regulation of cell metabolism The changes acquired by cancer cells that cause their unregulated proliferation and growth usually include both oncogenic pathways and inactivated tumor suppressor pathways 2O2) (30%) were obtained from Sigma (USA). ROS dyes H2DCFDA (5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester), JC-1  and MitoTracker Red were obtained from Invitrogen (USA). NAC were dissolved in the growth medium. PI was dissolved in water. DHE and JC-1 were dissolved in DMSO as a stock buffer.N-acetyl-L-cysteine (NAC), dihydroethidium (DHE), propidium iodide (PI) and hydrogen peroxide (H−/− cells were maintained in DMEM (high glucose) supplemented with 10% fetal bovine serum  and 50 IU penicillin/streptomycin , and MCF-10A cells were cultured in DMEM/F12 containing 5% horse serum , 20 ng/ml EGF , 0.5 mg/ml hydrocortisone , 100 ng/ml cholera toxin , 10 µg/ml insulin  and 50 IU penicillin/streptomycin  in a humidified atmosphere with 5% CO2 at 37°C.3T3/wt and 3T3/Rb4 cells suspended in top agarose solution (0.3%) were poured over bottomed agarose (0.6%) previously solidified in 6-well plates. Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C for weeks, and then colonies were counted.For soft agar assay, 104 cells were seeded into each well of the 6-well plates. Apoptosis assays were carried out based on the instruction from the Annexin V Apoptosis Kit (BD Biosciences). Briefly, cells were collected and washed twice with binding buffer containing 10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl, and then resuspended at a concentration of 1×106 cells/ml in binding buffer. Hundred microliters of the cell suspension was mixed with 5 µL of Annexin V-FITC (BD Biosciences) and 10 µL of propidium iodide (50 lg/ml stock) and incubated at room temperature for 15 min. Four hundred micro liters of binding buffer was added to each assay after the incubation and apoptotic cells were determined using a FACScan (BD Biosciences). Since NAC or H2O2-induced cell death in 3T3 cells is mostly apoptosis, total Annexin V-positive cells were used to determine the level of apoptosis.For the apoptosis assay, 25×102− and hydroxyl radicals 6 cells/ml). The fluorescence intensity of intracellular DCF  or DHE  was measured using FACScan .Intracellular ROS production was monitored by the permeable fluorescence dye, H2DCFDA and/or DHE. H2DCFDA can readily react with ROS to form the fluorescent product 2,7-dichlo-rofluorescein (DCF) 2 for 30 min. Cells were collected, resuspended in 0.5 ml of PBS for Flow assay. All the data analyses were performed using FlowJo analysis software, version 6.0 (Tree Star).The mitochondrial membrane potential (MMP) was monitored by the MMP probe JC-1 dye, which emits red florescence  under high MMP and green florescence (525 nm) under low MMP conditions 2, washed twice with warm medium, and then immediately analyzed under a Nikon Eclipse TE2000-U fluorescence microscope . The fluorescence intensity of images was analyzed with ImageJ software (1.47).After cells were grown in 6-well plates for 24 h, they were cultured in the complete medium containing 500 nM of MitoTracker Red for 30 min, at 37°C, 5% COWe used genetically-encoded Grx1-roGFP2 fusion protein to measure the redox state in cells The ratios of GSSG/GSH and NADP+/NADPH in 3T3 cells were measured using Glutathione (GSH/GSSG) Fluorometric Assay Kit and NADP/NADPH Quantitation Colorimetric Kit  according to the manual instruction.6 cells) was aliquoted in a 15 ml polypropylene tube and 3 ml of cold absolute ethanol was added dropwise while vortexing. Cells were fixed overnight at 4°C, washed twice with PBS, and then incubated with 1 ml of PI staining buffer  for 30 minutes at 37°C. Cells were analyzed using a FACScan . Cell cycle distribution was analyzed using FlowJo (6.0) software (Tree Star).Cells were seeded in 10-cm dishes for 48 h, trypsinized to single cells, and then washed twice with PBS. 1 ml of cells . Purified RNA was quantified by spectrophotometry. The mRNA levels of Bax, Bak, Bad and GAPDH were detected by standard RT-PCR ) using a random primer and then the specific primers as following:Mouse GAPDH-F: gcacagtcaaggccgagaatMouse GAPDH-R: gccttctccatggtggtgaaMouse Bax-F: aggatggctggggagacaccMouse Bax-R: ctgccacccggaagaagaccMouse Bak-F: cgctacgacacagagttccaMouse Bak-R: ggtagacgtacagggccagaMouse Bad-F: gcttagcccttttcgaggacMouse Bad-R: cccaccaggactggataatgGAPDH mRNA level was used as the normalization control.After desired treatments as specified as indicated, cells were washed twice with PBS and lysed in buffer . Equal amounts of protein (30 µg) were loaded onto 15% SDS-PAGE gels. Western detection was carried out using a Li-Cor Odyssey image reader . Anti-Rb, anti-Bad, anti-myc, anti-Ras and anti-β-Actin antibodies were obtained from Cell Signaling Technology , and were used with a dilution of 1∶1000. Anti-Bax, anti-Bak and anti-Bcl-XL antibodies were from Bioss (China), and were diluted by 1∶100. The goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG secondary antibodies were obtained from Li-Cor (USA). The final concentration of the secondary antibodies used was 0.1 µg/ml.Mitochondrial target sequence of human pyruvate dehydrogenase alpha 1 CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTshScramble: CCGGCGACGAGTCAAACAAGCCAATCTCGAGATTGGCTTGTTTGACTCGTCGTTTTTshRb Viral packaging was done according to a previously described protocol P<0.05 was considered as statistically significant.Unless otherwise indicated, at least three independent assays were carried out and the values are presented as mean ± S.D. Statistical significance was assessed by Student’s two-tailed t test and Drosophila−/− (Rb knockout) cells to investigate the involvement of Rb in the redox homeostasis. First, we used fluorescent dyes, H2DCFDA that mainly detects the hydroxyl radical −/− cells compared to 3T3/wt cells .Rb was implicated in the control of redox homeostasis in wt cells . Moreoveut cells . Therefo−/− cells  in 3T3 cells , an antioxidant, to reduce oxidative stress. Addition of NAC obviously reduced ROS levels  and incrT3 cells , and theT3 cells . MeantimT3 cells , and the−/− cells. RT-PCR results showed that mRNA levels of Bak and Bad, in particular Bad, were significantly enhanced in 3T3/Rb−/− cells compared to the wt cells . These observations suggest that the DCF fluorescence intensity in wt cells indeed represents the baseline. However, NAC apparently decreased the excitation ratio of 400/480 nm in wt cells like in Rb knockout cells (−/− cells unaffected and only slightly increased apoptotic cells upon the treatment with a high concentration of NAC (20 mM) were observed (2O2 increased ROS level in both wt and knockout cells (data not shown), and it also induced a more oxidative state in these cells , consistent with a previous report 2O2, RasV12 over-expression further increased the oxidative state in 3T3/Rb−/− cells , unlike antioxidant NAC that promoted colony formation of 3T3/Rb−/− cells  in both MCF-10A/shScr and MCF-10A/shRb cells, particularly in MCF-10A/shRb cells, the addition of NAC reduced ROS level in MCF-10A cells expressing shRb and/or RasV12  OS level . The oxi1-roGFP2 . Furtheroft agar . Unlike nockdown . The resoft agar , becauseoft agar . RasV12 r RasV12  as well r RasV12 . Accordi0A cells , the red0A cells . These d0A cells .−/− cells. Re-expression of Rb in 3T3/Rb−/− cells reduces the level of Bad, increases MMP and restores redox homeostasis. Since the expression mitochondria-targeted catalase and Bcl-XL also helps to maintain the redox homeostasis and MMP in 3T3/Rb−/− cells, redox stress appears to result from mitochondria. Antioxidant NAC that blocks ROS also enhances MMP, suggesting that the elevated level of ROS can exacerbates the loss in MMP. A small amount of ROS from mitochondria can stimulate ROS generation in cytoplasm, in turn, cytoplasmic ROS can target mitochondria to release more ROS Rb is a tumor suppressor, and regulates various biological progresses, such as cell proliferation, development, metabolism and cell death V12-mediated transformation in 3T3 cells V12 also depends on the redox state regulated by Rb status or NAC.Rb controls cell cycle and proliferation, and it is also required for Ras−/− cells are more sensitive to oxidative stress but more tolerant towards NAC treatment in contrast to 3T3/wt cells that are sensitive to NAC treatment but not oxidative induction. At the same time, we observe that the addition of NAC leads to more reductive redox while H2O2 induces more oxidative redox in both 3T3/wt and 3T3/Rb−/− cells compared to the control treatments. NAC only decreases ROS level in 3T3/Rb−/− cells but leaves ROS level unaffected in 3T3/wt cells that almost do not have basal ROS. These data suggest that it is redox state but not ROS level that determines the fate of 3T3 cells and it appears that only the 3T3 cells with a proper redox state can survive (−/− cells is more oxidative in contrast to that in 3T3/wt cells, and this difference leads to their distinct sensitivity to NAC and H2O2 treatments.3T3/Rb survive . The basV12 further increases the oxidative redox state in MCF-10A cells, and thus it contrarily inhibits the transformation of MCF-10A/shRb cells although it transforms MCF-10A/wt cells. These observations also support another possibility that cells with some proper redox state can be transformed while Rb is just a regulator of redox homeostasis (Rb deletion is able to confer the transformation ability in 3T3 cells given the redox state controlled by NAC or GSR over-expression. This suggests that Rb inactivation has the potential to transform 3T3 cells but such ability is blocked by the over-oxidative redox state. Indeed, Rb knockdown in MCF-10A cells induces oxidative redox state and transforms these cells. This inconsistency possibly results from the fact that Rb knockdown is unable to completely block Rb expression like Rb knockout and thus does not induce an over-oxidative state in MCF-10A cells. Raseostasis . Indeed,−/− cells have a more oxidative redox state and grows faster compared to 3T3/wt cells, but both do not grow in soft agar. However, increasing oxidative state in 3T3/wt cells or decreasing oxidative state in 3T3/Rb−/− cells transform both of cell lines. These observations support the idea that different redox states in 3T3 cells determine the corresponding biological activities (Over-oxidative or over-reductive redox state leads to death in 3T3 cells. 3T3/Rbtivities . Cells wROS is involved in diverse biological progresses and tightly associated with redox homeostasis Our results suggest that critical regulators involved in maintaining redox homeostasis could be selective targets for cancer cells. It will be of interest to further investigate the generality that redox homeostasis determines the fate of cells and the underlying mechanism. Since the redox homeostasis in cells is maintained by many components, such as glutathione system and thioredoxin system, the development of sensitive biosensors to detect in real-time the redox potential in cells is strongly desired. The quantitative analysis between redox potential and the biological activities of cells may reveal some aspect of the nature of life.