Background: Nutrients stimulate calcium dependent activation of energy metabolism, in pancreatic beta cells.Results: Glucose-induced ATP synthase-dependent respiration is strictly calcium-dependent, with little or no effect of calcium on the NAD(P)H response.Conclusion: Calcium coordinates oxidative metabolism and respiration in pancreatic beta cells.Significance: Calcium has novel mitochondrial targets downstream of mitochondrial dehydrogenases. + ratio.Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P) An increased energy demand, as observed during stimulation of a cell, enhances ATP hydrolysis that must be compensated for by accelerated ATP synthesis. For example in the heart, mitochondrial respiration can adjust over a wide range matching the enhanced work load associated with an increase in beating frequency. Interestingly, energy intermediates such as ATP, ADP, and phosphocreatine remain unchanged even when the heart frequency is augmented severalfold , and mitochondria are, therefore, crucial to maintain a high ATP/ADP P0%, and meralfold . Likewiseralfold , respiraeralfold , 5, and eralfold  to match,2+ channels open, and associated cytosolic Ca2+ rises trigger insulin granule exocytosis.In pancreatic beta cells, the rise in the cytosolic ATP/ADP Pi). Both the electrical and the chemical proton gradient across the inner mitochondrial membrane are elevated after stimulation of beta cells with nutrients H/NAD(P)+ ratio as demonstrated in neurons and cardiomyocytes  is coupled to the synthesis of ATP from ADP and inorganic phosphate H response to glucose in this cell type H levels.Here we have studied the mechanism resulting in enhanced energy metabolism in pancreatic beta cells during glucose stimulation. We observe a pronounced glucose-induced activation of ATP synthase-dependent respiration in INS-1E insulinoma cells and human islets. Activation of ATP synthase-dependent respiration but not the NAD(P)H response is potentiated by Cacyto pcDNA3 (cyto under the control of the rat insulin promoter (Ad-RIP-YC3.6cyto) was a kind gift of Prof. C. B. Wollheim (University of Geneva).Chemicals were from Sigma, Invitrogen, or VWR unless otherwise indicated. The YC3.6o pcDNA3  and 4mtDo pcDNA3  were kin2) in RPMI 1640 medium (Invitrogen) containing 11 mm glucose, supplemented with 10 mm Hepes (pH 7.3), 10% (v/v) heat-inactivated fetal calf serum , 1 mm sodium pyruvate, 50 μm β-mercaptoethanol, 50 μg/ml penicillin, and 100 μg/ml streptomycin.INS-1E cells were cultured at 37 °C in a humidified atmosphere (5% CO2) in RPMI 1640 medium complemented with 5.55 mm glucose, 10% (v/v) FCS (Brunschwig AG), 10 mm HEPES (pH 7.3), 1 mm sodium pyruvate, 50 μm β-mercaptoethanol, 50 μg/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml gentamycin (islet medium).Human islets from non-diabetic deceased donors were purchased from Tebu-bio. Donors had consented to donate organs for medical research. The islets were cultured at 37 °C in a humidified atmosphere (5% CO2) in DMEM containing 5.55 mm glucose supplemented with 10% (v/v) FCS (Invitrogen), 50 μg/ml penicillin, and 100 μg/ml streptomycin. Once cells reached 80% confluency, the culture medium was changed to the same medium lacking serum. After 2 days, the culture medium containing the matrix was collected and stored at −20 °C. Glass coverslips or Seahorse tissue culture plates were coated overnight with 804G matrix.The 804G bladder carcinoma cell line  and prot2+ was measured with the genetically encoded cameleon sensors YC3.6cyto or 4mtD3cpv. INS-1E cells were plated on polyornithine-treated 35-mm-diameter glass-bottom dishes (MatTek) and transfected with pcDNA3 vector carrying the Ca2+ sensor using Lipofectamine 2000 transfection reagent (Invitrogen). Two days after transfection cells were washed 4 times, and experiments were performed at 37 °C in Krebs-Ringer bicarbonate Hepes buffer (KRBH) containing 140 mm NaCl, 3.6 mm KCl, 0.5 mm NaH2PO4, 0.5 mm MgSO4, 1.5 mm CaCl2, 10 mm Hepes, and 5 mm NaHCO3 (pH 7.4). Glass coverslips were inserted in a thermostatic chamber (Life Imaging Services). The cells were preincubated for >15 min in KRBH basal (2.5 mm) glucose before recording. Cells were imaged on a DMI6000 B inverted fluorescence microscope using a HCX PL APO 63×/1.40–0.60 NA oil immersion objective (Leica Microsystems) and an Evolve 512 back-illuminated CCD with 16 × 16 pixels camera . Cells were excited at 430 nm through a BP436/20 filter. The two emission images were acquired with BP480/40 and BP535/30 emission filters. Fluorescence ratios were calculated in MetaFluor 7.0 (Meta Imaging Series) and analyzed in Excel (Microsoft) and GraphPad Prism 5 (GraphPad). Images were taken every 2 s.Cytosolic or mitochondrial Cacyto. Experiments were performed 1–2 days later as described for INS1E-cells. The islets were incubated under resting conditions (KRBH 1 mm glucose) for >15 min before initiating the experiment.Human islets were infected for 90 min at 37 °C with Ad-RIP-YC3.6m glucose. After 30 min of preincubation, in KRBH 1 mm glucose, the islets were either maintained in buffer containing 1 mm glucose, or the glucose concentration was raised to 16.7 mm for an additional 30 min. Insulin secretion is expressed as percent of content and was measured as described previously , rotenone (1 μm), and antimycin A (1 μg/ml). ATP synthase-dependent respiration was calculated as the difference in respiration rate before and after the addition of oligomycin. Other mitochondrial respiration (ATP synthase independent) was obtained after subtracting oligomycin-dependent respiration from the total mitochondrial respiration, which corresponds to the difference in respiration rate before and after the addition of rotenone plus antimycin A. Total human islet protein was quantified using a bicinchoninic acid-based protein assay .Oxygen consumption in INS-1E cells or human islets was measured using a XF96 instrument (Seahorse Biosciences). INS-1E cells were seeded into polyornithine-coated Seahorse tissue culture plates at a density of 20,000 cells per well. Two days later the cells were washed 2 times in KRBH 2.5 mm Ca2+ or a KRBH lacking Ca2+ but including 0.4 mm EGTA. Where the contribution of ATP synthase was assessed, oligomycin (2.5 μg/ml) was added during the last 10 min of the 16.7 mm glucose incubation.ADP and ATP were measured enzymatically as described . Cell exINS-1E cells or human islets were allowed to adhere to MatTek dishes coated with polyornithine or 804G matrix, respectively. All experiments were performed in KRBH buffer. A laser-scanning confocal microscope  with a HCX IRAPO L 25×/0.95 water objective was utilized to monitor NAD(P)H autofluorescence at 37 °C (Life Imaging Services).½.Laser scans at 727 nm  were used for two-photon excitation of NAD(P)H. Each 512 × 512 pixel image represents an average of 16 scans taken with a resonant scanner at 8000 Hz. The NAD(P)H emission band was collected at a 445–495-nm wavelength every 20 s. The excitation power of the laser was set to avoid cellular photo-damage. Data were processed with LAS AF software (Leica) and analyzed in Excel (Microsoft) and GraphPad Prism 5 (GraphPad). To normalize the NAD(P)H responses experiments were concluded by hydrogen peroxide addition, resulting in the almost complete loss of autofluorescence . Data were normalized to both base line = 1 and a minimal value (hydrogen peroxide) = 0. Alternatively, rotenone was used for standardization. The complex I inhibitor increases the NAD(P)H signal to the point where autofluorescence is likely close to maximal. NAD(P)H responses were normalized as a fraction between basal (set to 0) and maximal signal after rotenone (set to 1). The change in fluorescence (Δ NAD(P)H) was calculated as the difference between base-line value and the effect of glucose after reaching a plateau. In addition, data were fitted during stimulation with a one-phase exponential association equation to extract τn is the number of independent experiments. p values were obtained by Student's t test.Values are given as the mean ± S.E. 2+ signals in INS-1E cells (A–E) and human islet beta cells . In INS-1E cells the glucose-induced Ca2+ rise was preceded by a transient reduction of cytosolic Ca2+ followed by an initial Ca2+ overshoot before the cytosolic Ca2+ concentrations reached an apparent new steady state (B). At the single-cell levels the cytosolic Ca2+ responses to glucose were quite complex and variable (for examples see C–E). At basal glucose concentrations, Ca2+ transients were usually absent or of small amplitude. After glucose (16.7 mm) stimulation, the amplitude and/or the frequency of the cytosolic Ca2+ signals increased (C–E). In INS-1E and human islets it takes on average 73 ± 13 and 51 ± 14 s, respectively, before glucose-induced Ca2+ transients are initiated. Ca2+ transients depend on plasma membrane depolarization initiated by the closure of KATP channels. The KATP channel opener diazoxide, therefore, hyperpolarizes beta cells and lowered cytosolic Ca2+ below basal levels (C). Mitochondria act upstream of this process by controlling the cellular ATP/ADP ratio. Inhibition of respiration, therefore, blocks insulin secretion by interrupting the triggering pathway (2+ transients (D). The inhibition of Ca2+ transient was almost complete and very rapid (93.5 ± 75 s). Usually, only 1–2 more transients were observed after blocking respiration. Oligomycin blocked mitochondrial ATP synthesis but only partially inhibited respiration. Nevertheless, oligomycin arrested cytosolic Ca2+ transients over a similar time-course as rotenone and antimycin A (E).Glucose metabolism initiated cytosolic Ca1E cells , A–E anddy state B. At thencreased , C–E. Inl levels C. Mitoch pathway , 27. Theansients D. The inimycin A E.2+ rises are translated into mitochondria. In the matrix Ca2+ signals can be studied using 4mtD3cpv, a calcium probe that is efficiently targeted to the mitochondria of INS-1E cells (F). Glucose rapidly induced a large mitochondrial Ca2+ transient before a new steady state was reached (G). At the single cell level the mitochondrial Ca2+ responses were quite variable (H and I) but were frequently initiated by a larger mitochondrial Ca2+ transient (H). Preventing mitochondrial ATP synthesis using oligomycin ended mitochondrial Ca2+ spikes, and the matrix Ca2+ concentration returned to basal levels (I). Oligomycin, therefore, ended glucose-induced Ca2+ rises in both the cytosolic and mitochondrial compartment, causing a pronounced reduction of glucose-stimulated insulin secretion (L).Glucose-induced Ca1E cells F. Glucos reached G. At thevariable , H and Iransient H. Prevenl levels I. Oligomecretion L.2+ signaling (B-D). The small cytosolic Ca2+ transients that can be observed at 16.7 mm glucose ceased shortly after blocking ATP synthesis (C and D), and the net cytosolic Ca2+ concentrations returned to base line within minutes after inhibition of ATP synthase (285 ± 29 s). Consistent with this analysis at the single cell level, the average response shows that shortly after blocking the ATP synthase, the Ca2+ signal started to decline (B). Within several minutes cytosolic Ca2+ returned to basal levels corresponding to the signal before glucose stimulation (A and B). Via its effect on Ca2+ signaling, inhibition of mitochondrial ATP synthesis also caused a reduction of glucose-stimulated insulin secretion (E). These results are consistent with human islet perifusion experiments where azide rapidly lowered insulin secretion ) was considered to be non-mitochondrial and was not further studied. Blocking ATP synthase in glucose-stimulated INS-1E cells using oligomycin caused a rapid but only partial inhibition of respiration. We refer to this fraction as ATP synthase-dependent (oligomycin-sensitive) respiration, which is responsible for about 40% of total mitochondrial respiration in INS-1E cells . The larger fraction (60%) is mitochondrial respiration not directly linked to ATP synthesis .The respiratory response of INS-1E cells to glucose can be divided into two phases: an immediate acceleration of respiration occurring during the first 5 min and a subsequent slower but continuous acceleration of respiration until a new steady state was reached after 40 min of glucose stimulation . Human islet data were expressed relative to basal respiration before glucose stimulation. This corrects for the variability of respiration rates between wells due to differences in the number of attached human islets (A and B). Glucose-induced respiration increased rather rapidly as a new steady-state of oxygen consumption was reached within 12–18 min (A). Oligomycin caused a marked reduction of respiration in glucose-stimulated human islets. Rotenone and antimycin A further reduced respiration as expected. In glucose-stimulated human islets 75 ± 7% of total mitochondrial respiration was oligomycin-sensitive. The observed contribution of ATP synthase activity to respiration was, therefore, much larger than in INS-1E cells (A). Consequently, in human islets ATP synthase-independent respiration was smaller (25 ± 7%).Respiration of human islets was also enhanced after glucose stimulation A. Human n islets , A and B2–18 min A. Oligom1E cells A. ConseqA and B) and human islets (A). In resting INS-1E cells (2.5 mm glucose), oligomycin-sensitive respiration was almost absent (B and E). We conclude that the observed glucose-induced acceleration is mainly due to activation of ATP synthase-dependent respiration. Most of this glucose effect is reversed after oligomycin addition (A). Consistent with this interpretation, oligomycin-independent respiration was only slightly lower in INS-1E cells maintained in basal conditions (2.5 mm) compared with 16.7 mm glucose (F).Comparison of oligomycin-sensitive and -insensitive respiration revealed that glucose specifically activated ATP synthase-dependent respiration both in INS-1E cells , A and Bn islets A. In rest absent , B and Eaddition A. Consis glucose F.A). However, after glucose stimulation of human islets ATP synthase-dependent respiration was increased 1.9-fold (A and C). The ATP synthase-independent fraction on the other hand was not significantly affected by glucose (A and D). We conclude that glucose notably stimulates ATP synthase activity (oligomycin-dependent respiration), having little or no impact on other mitochondrial respiration-dependent processes in INS-1E and human beta cells.In human islets oligomycin also inhibited respiration under basal glucose conditions A. Howeve1.9-fold , A and C glucose , A and D2+ signals are required for the stimulation of mitochondrial respiration , cytosolic and mitochondrial Ca2+ signals in INS-1E cells were completely suppressed and glucose-induced respiration was strongly blunted . In the absence of Ca2+ signaling, glucose stimulated respiration initially, but thereafter respiration rates remained close to constant . Under Ca2+-free conditions, ATP synthase (oligomycin)-dependent respiration was 2.5-fold lower than in control glucose-stimulated INS-1E cells . Unexpectedly, in the absence of Ca2+, inhibition of respiration by oligomycin was transient . The reason for recovery of respiration after oligomycin treatment is not clear. Rotenone plus antimycin A reduced respiration efficiently starting, however, from lower initial values of respiration. As a consequence, ATP synthase-independent respiration was reduced by a factor of 1.8 when Ca2+ signaling was prevented .Glucose-induced Capiration , 17. We,2+ also had a very strong inhibitory effect on glucose-induced respiration (B and C). Oligomycin-sensitive respiration was reduced 2.1-fold in the absence of Ca2+ signaling (C), whereas oligomycin-insensitive respiration was not significantly affected (D). As Ca2+ is essential for insulin granule exocytosis, glucose-induced insulin secretion was completely suppressed when Ca2+ signaling was prevented (E). In the present system, it is therefore not possible to study the impact of Ca2+ control of mitochondrial energy metabolism on insulin secretion. In summary, we find that Ca2+ signals control ATP synthase-dependent respiration in INS-1E cells and human islets.In human islets the absence of extracellular Capiration , B and Cignaling C, whereaaffected D. As Ca2revented E. In the2+ signaling on ATP and ADP changes, we looked at a later time point . Blocking mitochondrial ATP synthesis with oligomycin lowered ATP but also increased ADP dramatically. Consequently, the ATP/ADP ratio was >70 times lower than in control glucose-stimulated cells (C). We conclude that in INS-1E cells, glycolysis is unable to maintain a physiological ATP/ADP ratio when mitochondrial ATP synthase is blocked. Preventing calcium signaling had no significant effect on the glucose-induced changes in ATP, ADP, or the corresponding ATP/ADP ratio  after glucose addition , 29. To  30 min;  as the rith time . The chaith time , A and Bed cells C. We conDP ratio . These r2+ to control mitochondrial metabolism and respiration has been mainly attributed to the activation of Ca2+-regulated dehydrogenases H can be used as a measure of the NAD(P)H/NAD(P)+ redox state and, therefore, indirectly as a read-out of oxidative metabolism (A). This is consistent with an earlier study finding 5–8-fold higher concentrations of NAD(P)H in mitochondria compared with the cytosol (The ability of Caogenases . This shtabolism . NAD(P)Htabolism A. This i cytosol . NAD(P)HB and E; Ref. 31–Fig. 6C). The net increase of the NAD(P)H signal varied strongly between different days of the experiment but averaged 53.8 ± 9.7% over basal . Lowering the glucose concentration rapidly decreased the NAD(P)H autofluorescence , which returned to basal levels or remained slightly elevated when compared with the signal measured before glucose addition.Glucose increased the steady-state NAD(P)H levels consistent with earlier studies (A) with a half-time of 121.2 ± 43.5 s . The net NAD(P)H fluorescence increase was 28.7 ± 11.7%  over basal. We conclude that in INS-1E cells and human islets, variations of the glucose concentration are very rapidly translated into changes of the NAD(P)H levels consistent with the role of the beta cell as a nutrient sensor.Similar experiments were performed averaging the NAD(P)H signal over individual islets . GlucoseFig. 7E). Complex I mediates the transfer of electrons from NADH, which oxidizes the co-factor to NAD+. Blocking complex I, therefore, leads to the accumulation of reduced NADH explaining the increased autofluorescence. A similar response was observed when blocking ATP synthase with oligomycin (H). Inhibition of ATP synthase slows upstream respiration and thereby NAD(P)H oxidation. Taken together, these results suggest a balance of NADH production (oxidative metabolism) and NADH oxidation (respiratory chain activity) that determines NAD(P)H steady-state levels.Once NAD(P)H signals had reached a steady state after glucose stimulation, the complex I inhibitor rotenone could further elevate NAD(P)H levels E. Compleigomycin H. Inhibi2+ signals on NAD(P)H, glucose responses were repeated in Ca2+-free KRBH containing 0.4 mm EGTA. Removal of Ca2+ under basal glucose conditions (2.5 mm) caused an immediate small reduction of the NAD(P)H signal in INS-1E cells (B). Although cytosolic Ca2+ signals are weak under resting conditions, completely blocking Ca2+ influx appears to affect the NAD(P)H steady-state levels consistent with an impact of Ca2+ on mitochondrial oxidative metabolism. Surprisingly, when raising glucose in the absence of Ca2+, NAD(P)H levels increased as rapidly as under control conditions . Furthermore, although the starting level of the NAD(P)H signal was lower, the final steady-state NAD(P)H levels were not significantly different when Ca2+ was omitted (B and D). Furthermore, re-addition of Ca2+ after glucose stimulation in Ca2+-free medium did not further increase the NAD(P)H steady-state level . This lack of calcium dependence was surprising especially given the striking Ca2+ effect on respiration (A and C). To better quantify the observed NAD(P)H responses to glucose, we used an alternative calibration strategy. As demonstrated in Fig. 6E, rotenone, which blocks complex I, increased the NAD(P)H signal to the point where autofluorescence was likely close to maximal. Rotenone was chosen over other inhibitors of the respiratory chain (oligomycin or cyanide) as it gave the most robust and reproducible increase of NAD(P)H signals in INS-1E cells (data not shown). Using this calibration, NAD(P)H responses were expressed as a fraction between basal (set to 0) and maximal signal after rotenone (set to 1). Glucose stimulation of INS-1E cells raised the NAD(P)H signal to 0.53 of maximal. When removing extracellular Ca2+ the NAD(P)H signal decreased below basal but reached levels not significantly different from the control after glucose stimulation (0.56). We conclude that steady-state NAD(P)H levels are mainly determined by the prevailing stimulatory glucose concentration with little further influence of Ca2+ signaling.To assess the effect of Ca1E cells B. Althoupiration , A and C2+ removal by EGTA. Islets NAD(P)H signals became unstable possibly due to altered binding of the islet to the matrix-coated glass coverslips when Ca2+ was chelated. We, therefore, used a combination of Ca2+ channel blockers isradipine, ω-agatoxin. and NNC 55-0396, which were shown previously to almost completely prevent voltage-dependent Ca2+ uptake in human beta cells . By blocking Ca2+ influx the three inhibitors also prevented glucose-induced insulin secretion (F). In addition, the Ca2+ channel blockers reduced the NAD(P)H signal in human islets when incubated under resting glucose conditions (A), similar to the effect of removing extracellular calcium from INS-1E cells (B). After stimulation with 16.7 mm glucose, NAD(P)H levels increased rapidly whether or not the Ca2+ channel inhibitors were present (B). Furthermore, the steady-state NAD(P)H signals reached after glucose (16.7 mm) stimulation were similar when the islets were pretreated with the Ca2+ channel inhibitors (C). We cannot, however, exclude possible smaller differences as net NAD(P)H increases varied strongly between individual islets. To address this issue, we stimulated human islets with glucose first to add the inhibitors of voltage-gated Ca2+ channels once the NAD(P)H signal had already reached a new steady state (E). Under these conditions, blocking Ca2+ influx had little or no effect on the NAD(P)H signal. We conclude that the glucose-induced NAD(P)H levels in beta cells in contrast to the respiratory rate is not strongly influenced by Ca2+ signaling.In human islets we failed to assess the effect of Cata cells . The effaddition D. By bloecretion F. In addnditions A, simila1E cells B. After  present B. Furthehibitors C. We candy state E. Under B). It is, therefore, unlikely that the ADP concentration is the sole regulator of mitochondrial respiration and ATP synthesis in INS-1E cells.Mitochondria are able to adjust their ATP synthesis to the energy requirement of the cell. Depending on the biological process, such adjustments must be rapid to avoid a drop of the phosphorylation potential. The mechanisms underlying such control of oxidative phosphorylation remain poorly understood. Early work with isolated mitochondria suggested that mitochondria mainly work on demand, enhancing respiration as a response to an increase in extramitochondrial ADP. In several cell types this control mechanism is not likely of relevance as it was shown that during physiological cell activation the phosphorylation potential does not decrease, and ADP levels are not elevated , 8. In tFig. 5KATP channel, which initiates the downstream events leading to insulin secretion. This may be accomplished through the increased provision of nutrient-derived metabolites for mitochondrial metabolism. The term “glucose push” has been coined to describe this initial activation of beta cell mitochondria by glycolysis-derived pyruvate . These results point to a special characteristic of beta cell energy metabolism. Rapid NADH provision for respiration is able to accelerate respiration over a very short time-course, which is achieved despite decreasing ADP levels. This tight link between glucose concentration and NAD(P)H may allow the beta cell to rapidly increase the ATP/ADP ratio necessary to induce electrical activity. Here we have studied the activation of beta cell mitochondria during glucose stimulation focusing on oxidative metabolism (following the NAD(P)H autofluorescence signal) and ATP synthase (oligomycin)-dependent respiration. Respiration is essential to maintain beta cell secretory activity. Inhibiting respiratory chain complexes blocks glucose-induced insulin secretion  similar to the combination of the complex I and III inhibitor. Glucose-induced mitochondrial Ca2+ signals were also suppressed when blocking ATP synthesis in INS-1E cells. The driving force for Ca2+ uptake into mitochondria is normal or even elevated as inhibition of ATP synthase by oligomycin hyperpolarizes the inner mitochondrial membrane  respiration is the main component of mitochondrial respiration –42. Our –2+. The high rates of uncoupling (depolarizing) may also help beta cells to maintain a normal membrane potential despite inhibition of ATP synthase (hyperpolarizing effect) under resting glucose conditions. Uncoupling slows ATP synthesis and, therefore, helps to prevent electrical activity in resting beta cells. Why this is mechanism is further exaggerated in INS-1E cells is not clear.In agreement with our data, prominent uncoupling has previously been observed in INS-1E cells  as well Glucose stimulation caused a marked acceleration of respiration in INS-1E cells consistent with earlier reports , 37. We After the stimulation of beta cells, both the electrical and chemical component of the mitochondrial membrane potential, increasing the driving force on the ATP synthase , 9. This2+ rises H rise. To block Ca2+ signaling in human islets, we used a combination of Ca2+ channel blockers H was unaffected. These results are further supported by kinetic data, which show that the NAD(P)H rise precedes the glucose-induced Ca2+ signals H in resting beta cells consistent with the possibility that Ca2+ signals do have an impact on the NAD(P)H redox balance when substrate levels are limiting. Taken together our findings demonstrate that the initial activation of beta cell mitochondria by glucose drives NAD(P)H formation without a need for Ca2+ signaling.Mitochondrial activation depends on mitochondrial matrix Ca2+ rises , 15. Theogenases . To addrblockers . In the  signals , and 7 c signals , 33. We 2+ signals were prevented both in INS-1E cells and human islets (C and 4B). Also, from a kinetic point of view, NAD(P)H and respiratory responses were clearly distinct. After the rapid glucose-induced response, the NAD(P)H signal remained elevated as long as the stimulatory glucose concentrations was maintained. Respiration augments rapidly early after glucose stimulation and continues to increase at later time points when NAD(P)H has already reached a new steady state.In contrast, the glucose-induced respiratory response was almost completely suppressed when Can islets C and 4B.2+-sensitive dehydrogenases) and respiration (rate-limiting complex of the respiratory chain or ATP synthase). The initial rapid NAD(P)H increase depends on glucose push. After this early response, Ca2+ continuously activates dehydrogenases to maintain NAD(P)H at this elevated level. Although the dehydrogenases produce more NADH per time during this second phase, no further net increase of the NAD(P)H signal was observed. This is due to the accelerated respiratory chain activity, which assures rapid re-oxidation. Such coordinated activation of dehydrogenases and oxidative phosphorylation allows a net increase in respiration without further affecting the NAD(P)H/NAD(P)+ ratio. Our population experiments do not exclude the possibility that at the single cell level there are Ca2+-dependent NAD(P)H transients as observed in a number of cell types previously H levels observed after glucose stimulation were in an equilibrium that could be shifted to a more oxidized or reduced state. Removal of substrate rapidly lowered the NAD(P)H signal, whereas inhibition of respiration with either rotenone or oligomycin caused a rapid increase of the NAD(P)H autofluorescence. Coordinated regulation of NAD(P)H formation and oxidation implies that Catabolism , 46.2+ signaling linked to cell activation is required to increase ATP synthase-dependent respiration for full activation of beta cell mitochondria. This activation occurs gradually over 12–18 min in human islets during which oxidative metabolism and respiration are increased in a coordinated manner H levels by providing elevated concentrations of the mitochondrial substrate pyruvate. Subsequently, Cad manner . As a re


RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.in vitro and tumorigenesis assay in vivo.Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines  and non-HCC cell lines  were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC. Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death in vivo animal studies have demonstrated the success and feasibility of RNAi for the treatment of various diseases In recent years RNA interference (RNAi) has emerged as a potential treatment modality for cancer therapy. RNAi is a sequence-specific posttranscriptional gene silencing process. Since its discovery, RNAi has been rapidly developed into a powerful technique for both therapeutic and gene functional research. Several clinical trials published so far and numberous in vivo cellular uptake and intracellular delivery still remain a major challenge for therapeutic application of RNAi in vitro and in vivoHowever, although RNAi is highly attractive as a therapeutic approach, several hurdles must be overcome before RNAi can be successfully introduced into the clinic. These include efficiency of cellular uptake, specific guidance to target tissue or cell, and sustained efficacy in vitro and in vivo. The feasibility of the system as new agent against HCC was tested based on the finding in previous research that silencing Atg5 gene could enhance sorafenib lethality in vitro and in vivo.The purpose of this study was to develop an efficient HCC tissue-specific RNAi system and investigate the possibility of the system for HCC treatment. We constructed two different HCC tissue-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by an AFP promoter (AFP-miRNA system and AFP-Cre/LoxP-shRNA system). The effectiveness and tissue-specificity of the systems were examined in HCC and non-HCC cells ATCC), HCCLM3 Human HCC cell lines Hep3B, HepG2, PLC/PRF/5 (A SV40-hAFP promoter from pDrive-SV40-hAFP (InvivoGene) was chosen as the AFP promoter. The activity and tissue-specificity of the AFP promoter was assayed using GFP reporter in AFP-producing and non-AFP-producing cell lines. A recombinant lentivirus vector containing the AFP promoter and GFP reporter gene was constructed as follow. The AFP promoter was synthesized according to the instruction of the pDrive-SV40-hAFP with adding NheI and EcoRI restriction enzyme cutting sites (RECS) See . After aThe AFP-miRNA targeting Beclin 1 was constructed as follow. Four miRNAs targeting Beclin 1 with negative control were designed and constructed into pcDNA6.2-GW/EmGFP-miR (Invitrogen). The vectors were named as pcDNA-miR-1, pcDNA-miR-2, pcDNA-miR-3, pcDNA-miR-4 and pcDNA-miR-NC. Then the 900 bp CMV-miRNA-EGFP structure was amplified from the templates pcDNA-miR-1/2/3/4/NC by PCR using primers Gecp-1,2 . Then thTGCTGTAATGGAGCTGTGAGTTCCTGGTTTTGGCCACTGACTGACCAGGAACTCAGCTCCATTA; Beclin 1-R: CCTGTAATGGAGCTGAGTTCCTGGTCAGTCAGTGGCCAAAACCAGGAACTCACAGCTCCATTAC.The miRNA sequence: Beclin 1-F: (1) AFP-Cre vector: The pCDH-CMV-MCS-EF1-Puro (SBI) was used to generate the AFP-Cre. The AFP promoter fragment was amplified from the template AFP-GFP by PCR using primer Gecp-7,8 ((2) LoxP-shRNA vector: Fragment A and fragment B were cloned from template pSil02 vector  by PCR using primers Gecp-11,12 and Gecp-13,14 (in vitro and in vivo. (1)In vitro evaluation: The AFP-producing HCC cells (HCCLM3/HepG2/Hep3B) and non-AFP-producing cells (L-02/Hela/SW1116) were infected with the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) . The cells were observed under fluorescence microscope every day. Q-PCR and western blot analysis of Beclin 1 gene silencing were performed one week later. (2)In vivo evaluation: Xenograft nude mouse model of HCC via orthotopic implantation and subcutaneous inoculation of HCCLM3 cells were established as previously described Intravenous injection: Twelve mice were randomly divided into two groups (n = 6 per group). One week after orthotopic implantation, the AFP-Cre/LoxP-shRNA system was intravenously injected via tail vein . The mice were sacrificed two weeks later. Intratumoral injection: Thirty-six mice were randomly divided into four groups (n = 6 per group). When the subcutaneous tumors reached a size of about 4 mm maximal diameter, the AFP-Cre/LoxP-shRNA system was intratumorally injected . All mice were sacrificed two weeks later. All of the tumors were resected. The frozen sections of the tumors were stained with DAPI (Invitrogen) and observed under fluorescence microscope as mentioned below. Q-PCR and Western blot analysis of Beclin 1 silencing were performed.The AFP-Cre/LoxP-shRNA system consists of AFP-Cre and LoxP-shRNA lentiviral vectors. The AFP-Cre vector contains a Cre gene driven by the AFP promoter. The LoxP-shRNA vector has a modified U6 promoter followed by shRNA against target gene (Beclin 1). The modified U6 promoter harbors a LoxP-CMV-eGFP-LoxP inside. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter will drive the expression of Cre recombinase which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates the U6 promoter. The activated U6 promoter then drives the down-stream shRNA expression and silences the target gene. The CMV-eGFP inside the U6 promoter serves as an indicator . The AFP-Cre and LoxP-shRNA vectors were constructed as follow: Gecp-7,8 . The CreGecp-7,8 . The AFPGecp-7,8 . The AFPGecp-7,8  was thencp-13,14 , respectcp-13,14 . A fragmcp-13,14  was genecp-13,14 . The fracp-13,14  was clonCCGGCCCGTGGAATGGAATGAGATTCTCGAGAATCTCATTCCATTCCACGGGTTTTTG;Beclin 1-R: AATTCAAAAACCCGTGGAATGGAATGAGATTCTCGAGAATCTCATTCCATTCCACGGG.The Beclin 1 shRNA sequence: Beclin 1-F: The quantitative real-time PCR (Q-PCR) analysis of Atg5 and Beclin 1 expression was performed as previously described Western blot analysis was performed as previously described Cell viability was determined with WST-8 cytotoxicity assay using Cell Counting Kit-8  as previously described After the mice were killed, the tumors were resected immediately and embedded in Tissue-Tek OCT compound using liquid nitrogen and stored at −80°C. Then the tissue samples were sectioned at 40 µm thickness with a cryostat. The tissue sections were stained with DAPI . Then they were observed and analyzed using confocal fluorescence microscope (Carl Zeiss).in vitro and in vivo. (1)In vitro evaluation: The MHCC97-L and PLC/PRF/5 cells were infected with the AFP-Cre/LoxP-shRNA-Atg5 . One week later, the virus-infected cells were exposed to sorafenib  for 24 h. Cells without infection and cells infected with AFP-Cre, LoxP-shRNA, or AFP-Cre/LoxP-shRNA-NC served as controls. Apoptosis was measured using Annexin V and PI flow cytometry as previously described (2)In vivo evaluation: Xenograft nude mouse model of HCC via subcutaneous inoculation of MHCC97-L cells (1×107) was established as previously described 8 TU AFP-Cre+0.5×108 TU LoxP-shRNA-Atg5 in 50 µl Opti-MEM). One week after AFP-Cre/LoxP-shRNA-Atg5 treatment, all mice in the Sorafenib group and Sorafenib+Cre/LoxP group were treated daily with sorafenib (30 mg/kg) via intraperitoneal injection. The animals were monitored every day and sacrificed 6 weeks after tumor implantation. All of the tumors were resected and the tumor weights were measured.A previously validated Atg5 shRNA See  was clonCCGGGCTAGCTGGCTGTCCATATTTCAAGAGAATATGGACAGCCAGCTAGCTTTTTTG;Atg5-R: AATTCAAAAAAGCTAGCTGGCTGTCCATATTCTCTTGAAATATGGACAGCCAGCTAGC.The Atg5 shRNA sequence: Atg5-F: t test between two groups or by one-way ANOVA for multiple groups. P values<0.05 were considered statistically significant.Statistical analysis was performed with SPSS 13.0 software . Results were presented as mean ± standard deviation (SD). Comparisons of quantitative data were analyzed using Student's A recombinant lentiviral vector containing GFP reporter gene driven by AFP promoter was constructed (AFP-GFP) . The actin vitro  targeting Beclin 1 gene was constructed . A CMV pin vitro . Q-PCR ain vitro . A MOI oin vitro . As compin vitro . The CMVin vitro . As highin vitro .As the AFP-miRNA required a high titer which was cytotoxic to target cells, it was not desirable for target therapy because it might cause damage to the adjacent normal cells and tissues. Therefore, another system (AFP-Cre/LoxP-shRNA) was constructed .in vivo. Systemic administration of the AFP-Cre/LoxP-shRNA system was firstlytested. Nude mouse model of HCC via orthotopic implantation was established using AFP-producing HCC cells (HCCLM3) and the AFP-Cre/LoxP-shRNA-Beclin1 was intravenously injected (in vivo (The AFP-Cre/LoxP-shRNA system was successfully established . The CMVinjected . Q-PCR ainjected . Intraveinjected . The ineinjected . The AFPinjected . Q-PCR a(in vivo .in vitro. Apoptosis assay showed that the apoptotic rate of cells (MHCC97L and PLC) treated with AFP-Cre/LoxP-shRNA-Atg5 plus sorafenibwas significantly higher than that of cells receiving sorefenib alone    . To furt in vivo . The ave= 0.039) . These rIn vitro and in vivo analysis showed that it could efficiently silence target gene in an HCC-specific manner. Application of the system targeting Atg5 was shown to be able to enhance sorafenib treatment in vitro and in vivo, suggesting its value for HCC therapy. Our system provides a usable tool for HCC tissue-specific RNAi therapy, which holds significant promise as new approach for the management of HCC.In this study, an efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. in vivo application as it posed threat to adjacent normal cells or tissues. The low efficacy of the AFP-miRNA was attributed to the weak activity of the AFP promoter. Although the AFP promoter was shown to able to drive GFP expression, the transcriptional activity appeared to be insufficient to mediate efficient miRNA expression. As the AFP-miRNA could not achieve satisfatory efficacy, we then attempted the shRNA strategy. The shRNA cannot be directly driven by the AFP promoter (Pol II) but it can be efficiently driven by Pol III promoter (such as U6). However, the Pol III promoters mediated RNAi cannot be controlled in a tissue-specific manner because the Pol III promoters are constitutively expressed in all cell types. To circumvent this problem, a switch structure (Cre/loxP system combined with a modified U6 promoter) was designed to obtain shRNA expression driven by AFP promoter. Our results strongly demonstrated that the novel system could efficiently mediate shRNA expression and specifically silence target gene of HCC cells or tissues. Further application of the system for HCC treatment showed that the system was valuable for HCC therapy. The system-mediated HCC-specific Atg5 silencing enhanced sorafenib treatment. Although the Atg5 gene was shown to be not an optimal target gene for HCC RNAi therapy as its efficacy was weak, the validation using Atg5 had demonstrated the value of the AFP-Cre/LoxP-shRNA system. With the application of new better target gene, a satisfactory effect of the system-mediated RNAi therapy will be achieved.In recent years, with tremendous advance in HCC research, increasing HCC-related genes have been discovered. Translating these findings into clinical therapy, especially target therapy, holds great promise for HCC treatment 9 TU/ml) and the nude mouse model (maximum volume of intravenous injection is 180 µl), the maximum intake is only 3.6×108 TU. Second, there is a considerable loss of the system during intravenous administration. The lentivrial vector used for this study has a wide host tropsin. It can quickly transfect any type of cells, although it works in only AFP-producing HCC cells. After being intravenously injected, it may immediately infect blood, endothelial cells or any other cells it encountered and then is considerably consumed before it reaches the tumor in the liver. The commonly used hydrodynamic injection was not used in this study. Although it is an effective way of delivering therapeutic agents into the liver in HCC mouse models Although the AFP-Cre-LoxP-shRNA system is shown efficient for HCC tissue-specific RNAi, it still carries some inherent disadvantages. The main one is that intravenous application of the system appears to be ineffective. This is not consistent with our expectations. The systemic administration of the system was thought to be able to achieve satisfactory efficacy since numerous studies had demonstrated that RNAi agents (siRNAs) could be easily delivered into the liver and liver tumor was suitable for intravenous application Taken together, in this study, an HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully developed. The AFP-Cre/LoxP-shRNA system can efficiently silence target gene in an HCC tissue-specific manner. The system mediated silencing of therapeutic gene is valuable as adjuvant therapy for HCC treatment. Our system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new therapeutic modality for the management of HCC.File S1The sequence of AFP promoter.(DOC)Click here for additional data file.File S2The AFP-Cre structure and U-LoxP-CMV-eGFP-LoxP-U6 structure.(DOC)Click here for additional data file.File S3Determination of the effective shRNAs targeting Beclin 1 and Atg5.(DOC)Click here for additional data file.File S4Comparison of the effects of AFP-Cre/LoxP-shRNA-Atg5 among sorafenib alone, and Cre/PoxP with and without sorafenib treatment.(DOC)Click here for additional data file.


We suppose that the up-regulation of stomatin by hypoxia and dex may enhance the barrier function of alveolar epithelia and mediate the adaptive role of GC to hypoxia.Stomatin is an important lipid raft-associated protein which interacts with membrane proteins and plays a role in the membrane organization. However, it is unknown whether it is involved in the response to hypoxia and glucocorticoid (GC) in alveolar epithelial cells (AEC). In this study we found that hypoxia and dexamethasone (dex), a synthetic GC not only up-regulated the expression of stomatin alone, but also imposed additive effect on the expression of stomatin in A549 cells, primary AEC and lung of rats. Then we investigated whether hypoxia and dex transcriptionally up-regulated the expression of stomatin by reporter gene assay, and found that dex, but not hypoxia could increase the activity of a stomatin promoter-driven reporter gene. Further deletion and mutational studies demonstrated that a GC response element (GRE) within the promoter region mainly contributed to the induction of stomatin by dex. Moreover, we found that hypoxia exposure did not affect membrane-associated actin, but decreased actin in cytoplasm in A549 cells. Inhibiting stomatin expression by stomatin siRNA significantly decreased dense of peripheral actin ring in hypoxia or dex treated A549 cells. Taken all together, these data indicated that dex and/or hypoxia significantly up-regulated the expression of stomatin  The mechanism of up-regulation of stomatin and the relationship between the expression of stomatin and membrane associated actin cytoskeleton in A549 cells were also investigated.Stomatin was first identified in human erythrocytes and absence of the protein is associated with a major abnormality in cation permeability in erythrocytes and a form of haemolytic anaemia known as overhydrated hereditary stomatocytosis (OHSt) 2 incubator. Rat AEC were isolated, purified and cultured as described previously 2, 5% CO2, 94% N2 for hypoxia exposure with or without dex treatment Human lung adenocarcinoma epithelial A549 cells were cultured routinely in DMEM/F-12 medium containing 10% newborn bovine serum  at 37°C in a 5% CO2 (a gas mixture of 8% O2 and 92% N2) for different times. After hypoxia exposure, animals were immediately anaesthetized and killed, and lungs were isolated for further experiments.The protocol for the study was approved by the Institutional Animal Care Committee of the Second Military Medical University in Shanghai, China. The animal care facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Sprague-Dawley rats, weighing from 200 to 250 g, were obtained from Shanghai SLAC laboratory animal company. All animals were acclimatized in our animal laboratory for at least 7 days before the experiment, being fed with standard laboratory chow and water. The randomly selected rats were put in a normobaric hypoxia chamber  and flushed with 8% Oet al. The animals were anaesthetized by intraperitoneal injection of 3% pentobarbital sodium (1 ml/kg BW), and adrenal glands were removed by the dorsal approach as described by Fleshner The adx rats were injected intramuscularly with 5 mg/kg body weight of dex  dissolved in 0.9% NaCl solution. Control adx rats were treated with 0.9% NaCl alone Total RNA was isolated using TRIzol reagent . Two micrograms of total RNA was reversely transcribed using reverse transcription reagents  in accordance with the manufacturer's instructions. Stomatin mRNA was analysed in triplicate using SYBR Green PCR Master Mix  on Mastercycler ep realplex . Primers used were: Stomatin (human) sense 5′-AGAGCTCCTGGTCCTCAA-3′, antisense 5′-TCTGTCCATCCAGCCAATG-3′. Stomatin (rat) sense 5′-CCCTGGCTGTGGCAAATA-3′, antisense 5′-GGGAAGACAATGGTGGAGT-3′. Amplification of stomatin cDNA was normalized to β-actin .The protein extracts from cells or tissues were electrophoresed on 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. After being blocked with 5% nonfat milk in tris-buffered saline tween-20 (TBST), the membranes were immune-blotted with a mouse anti-stomatin antibody  or a mouse anti-β-actin monoclonal antibody  overnight at 4°C followed by antimouse horseradish peroxidase conjugated secondary antibody . The blots were visualized by ECL reagent kit  following the manufacturer's recommended protocol and the relative expression of the protein bands of stomatin (31 kD) was quantified by scanning densitometry using β-actin as internal control.via PCR using the following primers, sense 5′-AGTGGGTACCAGGACTGTTTGTGACAA-3′,antisense 5′-GAAGATCTTCAGGAAGCAGCGGTGTCTG-3′. PCR reactions were performed with PrimeSTAR® HS DNA polymerase . The reaction condition was:one cycle 98°C for 1 min.; 30 cycles 98°C for 10 sec., 58°C for 15 sec., 72°C for 2 min. and 40 sec.; one cycle 72°C for 7 min. The amplification product was ligated to the blank luciferase reporter plasmid pGL3-Basic  via Acc65I and BglII restriction sites introduced by the primers. The constructed reporter plasmid containing about 2 kb (-1782 to +244) of promoter sequence of human stomatin gene was named STOM (-1782 to +244)-luc (STOM-1782) and the following truncated fragments containing the initial truncated position to +244 were named STOM-initial numbers separately, which were constructed using STOM-1782 as template and the forward primers were as follows:For cloning of stomatin promoter-driven reporter plasmid, genomic DNA was acquired from A549 cells using MagExtractor-Genome kit (Toyobo) according to the manufacturer's instruction. DNA sequence was amplified STOM-1630: 5′-TAGGGTACCAACAAGCACTGGCCTCGAG-3′, STOM-1246: 5′-ACGGGTACCACACCACCCCACCTGG-3′, STOM-288: 5′-TATCGGTACCCTATCCTCACCATCTGCTC-3′, STOM-162: 5′-TACAGGTACCAGCCCACAGAACTAGCG-3′, STOM-10: 5′-ATGTGGTACCTGGGTCTTGTGCCTGTG-3′. The sequences of all constructed plasmids were confirmed by sequence analysis. Deletion mutants whose the putative GR binding sites were deleted were made with the PrimeSTAR® HS DNA polymerase (Takara). To remove the five conserved base pairs in the putative GRE sequences, the following primers were employed. mGRE1, sense: 5′-CACGCCCCTCAGCCCACTAGCGGGAAGTGACTGCGA-3′, antisense: 5′-TCGCAGTCACTTCCCGCTAGTGGGCTGAGGGGCGTG-3′. mGRE2, sense: 5′-GCAGTCGCGCCGTGGAGGGAGGGCGGGGATTGGG-3′, antisense: 5′-CCCAATCCCCGCCCTCCCTCCACGGCGCGACTGC-3′. mGRE3, sense: 5′-CGGCAATCTGGGTCTTTCTGGCTCCTCAGGGCA-3′, antisense: 5′-TGCCCTGAGGAGCCAGAAAGACCCAGATTGCCG-3′. mGRE4, sense: 5′-CAGCATGGCCGAGAAGCGGCACGGGACTCCGAAG-3′, antisense: 5′-CTTCGGAGTCCCGTGCCGCTTCTCGGCCATGCTG-3′. mNF-κB, sense: 5′-TGCCTCTGGCTCCTCAGGCCCGGCGGCTCCGGGTTT-3′, antisense: 5′-AAACCCGGAGCCGCCGGGCCTGAGGAGCCAGAGGCA-3′. mAP-1, sense: 5′-GTGAGTCCCGCGTCCCCCTCCCCCGTGGACCGAGCC-3′, antisense: 5′-GGCTCGGTCCACGGGGGAGGGGGACGCGGGACTCAC-3′.Correct assembly of the deletion was confirmed by sequence analysis. For transient transfection reporter assay, A549 cells were seeded onto 48-well plates. Twenty-four hours later, cells were transiently transfected with the constructed promoter vectors (0.5 μg/well) using JetPEI  according to the manufacturer's instructions. Twenty-four hours later, cells were treated with dex for 24 hrs and lysed for detecting the reporter gene activity using dual luciferase assay kit (Promega).F-actin filaments in A549 cells were stained with 3 μg/ml molecular probes phalloidin-tetramethylrhodamine isothiocyanate (Sigma-Aldrich), mounted with the anti-fade reagent and then examined by a laser-scanning confocal microscope .et al. The expression vector encoding for human stomatin (pEGFP-N3-stom) and blank vector (pEGFP-N3) were constructed as described by Umlauf siRNAs to stomatin and the scrambled siRNAs were designed and obtained from GenePharma . The sequences for stomatin-siRNA were 5′-GUGGCGUUCUCAUUCUUAUTT-3′(sense), and 5′-AUAAGAAUGAGAACGCCACTT-3′ (antisense) respectively. A549 cells were transfected with a final concentration of 1 or 3 nM of siRNA using INTERFERin™ (PolyPlus-Transfection) for 12 hrs according to the manufacturer's instruction. Then the medium was changed and A549 cells were allowed to recover for 12 hrs before subsequent dex treatment or hypoxia exposure.anova in the SPSS13.0 software package. Differences were considered statistically significant at P < 0.05.Data were presented as mean ± S.E. and analysed using one-way 2) conditions for different times by RT PCR and Western blot. We found that hypoxia significantly up-regulated stomatin at mRNA and protein levels in a time-dependent fashion (P < 0.05).We first examined the expression of stomatin in A549 cells under normoxic or hypoxic (1% O fashion A and B. 2) for different times to determine if the effects of hypoxia on the expression of stomatin also occurred in vivo. As shown in P < 0.05).We further examined stomatin expression in the lung of rats after exposure to hypoxia (8% OP < 0.01). Ten nanomole dex also significantly increased stomatin expression at protein level in a time-dependent fashion (The effect of dex on stomatin expression in A549 cells was investigated using RT PCR and Western blot. Cells were treated with different concentrations of dex (0.1–100 nM) for 12 hrs, or 10 nM dex for different times. As shown in  fashion C. The st fashion D, indicaP < 0.01). Similar results were observed at protein levels in A549 cells  were removed to examine the effects of hypoxia on the expression of stomatin with or without supplying with dex (5 mg/kg). As shown in P<0.05) and in adx group . Moreover, administration of adx rats with dex for 12 hrs significantly increased the expression of stomatin mRNA (about 1.8-fold of that in the adx group) in normoxia, and further increased stomatin expression (about 3.7-fold of that in the adx group) in hypoxia. Similar results were observed at protein levels in different groups (in vitro and in vivo.It has been reported that t groups E. These P < 0.05), indicating that dex can up-regulate stomatin expression at transcriptional level.To determine whether hypoxia or dex regulates the stomatin gene at transcriptional level, a stomatin reporter plasmid STOM (-1782 to +244)-luc containing about 2 kb (-1782 to +244) of promoter sequence of human stomatin gene was constructed and transfected into A549 cells. The reporter activity was determined by luciferase assay after cells were exposed to hypoxia or treated with dex for different times. The results showed that reporter activity did not change obviously after cells were exposed to hypoxia A. HoweveTo determine the response elements which are responsible for dex induction, a series of 5′ deletion mutants of the stomatin promoter were constructed A and linThere were four potential GREs, one AP-1 and one NF-κB binding site within the −162 to +244 region of stomatin gene by transcription element search software analysis C. Using As actin cytoskeleton plays an important role in alveolar epithelial barrier and in many cellular functions, we examined the relationship between stomatin and actin cytoskeleton. We first studied the cellular localization of actin and stomatin in A549 cells transiently transfected with blank vector (pEGFP-N3) or EGFP-tagged stomatin expression vector (pEGFP-N3-stom). The results showed that EGFP-tagged stomatin protein was clustered in the membrane and around the perinucleus. Stomatin proteins in the membrane were co-localized with membrane-associated actin filaments labelled by rhodamine-phalloidine in A549 cells A.Then we investigated the effect of hypoxia or dex treatment on actin cytoskeleton for 12 hrs in A549 cells. As shown in We further inhibited the expression of stomatin in A549 cells with 1 or 3 nM stomatin small interfere RNA , was important for the effect of dex. As dex could result in a four- to fivefold increase in the levels of stomatin mRNA and the maximal induction of the activity of stomatin promoter reporter gene was only 2.8-fold, other GREs beyond this cloned promoter region may also contribute to the GR transcriptional regulation of stomatin In addition to hypoxia, we observed that dex also induced the expression of stomatin 2+-induced vesiculation in stomatin-deficient OHSt patients, and when stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased +, K+-ATPase, Na+ transporters, tight junction proteins and cell-cell adhesion molecules Recent studies have found that there existed membrane raft actin deficiency and altered CaAs a raft protein, stomatin is believed to combine several other membrane proteins besides actin, such as ASIC in sensory neuron in vitro and in vivo. The effects of dex occurred at transcriptional level. Up-regulated expression of stomatin by hypoxia and dex could stabilize membrane-associated actin cytoskeleton in A549 cells, which might enhance the barrier function of alveolar epithelia and mediate the adaptive role of GC to hypoxia.In summary, this study showed that hypoxia and dex significantly up-regulated the expression of stomatin individually or in combination


We also found that upon knockdown of endogenous wild-type p53 (p53-KD), MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H  or R261H  in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, β-catenin, and epithelial-to-mesenchymal transition (EMT) transcription factors  is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D) of Madin-Darby canine kidney (MDCK) cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules  Wild-type p53, a tumor suppressor, plays an important role in cell-fate determination through the regulation of the cell cycle, programmed cell death, differentiation or senescence ,2. Thus, Mutant p53 can form a heterotetramer with wild-type p53 and inhibit wild-type p53 to act as a tumor suppressor ,20. In aTo determine mutant p53 gain of function in morphogenesis and tumorigenesis, we examined mutant p53 activity using 3-D culture model of Madin-Darby canine kidney (MDCK) cells. MDCK cells in 3-D culture undergo a series of morphological changes and form polarized and growth-arrested cyst structures with hollow lumen, which re-differentiates into normal tubules upon induction by hepatocyte growth factor (HGF) ,31. HereBovine collagen solution (3.2 mg/mL) was purchased from Advanced Biomatrix . MEM medium and non-essential amino acid were purchased from Invitrogen . Recombinant human hepatocyte growth factor (HGF) was purchased from Sigma . TACCTCTCGCCAT-3′5′- GCAATC) was replaced with AACATCACGACAT-3′5′- GCAATC, was amplified with forward primer P1, 5′- AAGCTTACCATGCAAGAGCCACAGTCAGAG-3′, and reverse primer P2, 5′- CTCGAGCACATCTGTACCATGCAAAGT-3′. The resulting fragment encoding siRNA-resistant p53 was confirmed by sequencing and cloned into pcDNA4, and the resulting plasmid was designated as pcDNA4-siRNA-resistant mutant p53 , fragment 1 was amplified with forward primer P1, and reverse primer P5, 5′- GGCACAAACGTGTACCTCAAAGCTG-3′; fragment 2 was amplified with forward primer P6, 5′-TTGAGGTACACGTTTGTGCCTGTCC-3′, and reverse primer P2. Then, fragments 1–2 were mixed together as a template and amplified with primers P1 and P2. The resulting fragments encoding p53 (R163H) and p53 (R261H) were confirmed by sequencing and cloned into pcDNA4. To generated mutant p53-producing cell lines, pcDNA4-mutant p53 was transfected into MDCK cells. The resulting mutant p53-producing cell lines were selected with Zeocin and confirmed by Western blot analysis. To generate stable p53-KD cell lines with mutant p53 overexpression, pBabe-U6-siRNA was co-transfected with pcDNA4-mutant p53 into MDCK cells. The resulting cell lines were selected with puromycin and Zeocin. Both p53-KD and mutant p53 expression were confirmed by Western blot analysis and RT-PCR assay. To generate vectors expression a shRNA against canine p53 under the control of the U6 promoter, two 62-base oligos were annealed and then cloned into pBabe-U6 shRNA expression vector. The resulting plasmids were designed as pBabe-U6-shp53, which carries a puromycin selection marker . To genetant p53 . Next, t2. Culture medium was renewed every third day. For induction of tubulogenesis, culture medium plus 10 ng/mL of HGF was added to the culture plate.The MDCK cell line was obtained from American Type Culture Collection  and cultured in MEM medium supplemented with 10% fetal bovine serum and 1% non-essential amino acid. The overlay 3-D culture was carried out as described previously with some modifications . BrieflyWestern blot was performed as described . Antibod5′-GTGCCTCACAGAGTGCAAAA-3′, and antisense 5′-CCTGAATGTTGGGAGCATTT-3′. The Actin gene was chosen as a loading control and detected with primers 5´-ctgaagtaccccatcgagcacggca-3´ (sense) and 5´-ggatagcacagcctggatagcaacg-3´ (antisense).Total RNA was extracted from cells using TRIzol . cDNA was synthesized using M-MLV Reverse Transcriptase Kit  according to manufacturer’s protocol. The mRNA level of wild-type p53 was measured by PCR. The special primers to detect wild-type p53 are sense MDCK cells were cultured in a 6-well plate for ~12 d and then fixed with methanol/glacial acetic acid (7:1) followed by staining with 0.1% crystal violet. Experiments were conducted in triplicate.Cells were grown in a 6-well plate for 24 h. The monolayers were wounded by scraping with a P200 micropipette tip and washed two times with PBS. At specified time points after the scraping, cell migration was captured using phase contrast microscopy and cell monolayers were photographed using a Canon EOS 40D digital camera . Migration rate of cells was measured by averaging the time required to close the borders of cells. Six regions were analyzed in each well, and the result was expressed as the mean ± SD.t test. Values of P < 0.05 were considered significant.Data were presented as Mean ± SD. Statistical significance was determined by Student’s in vivo [MDCK cell line contains wild-type p53 and possesses the ability to form cyst structures when cultured in 3-D collagen gel . Upon inin vivo ,31. We sin vivo . In addiin vivo . Mutation of p53 is a frequent event in renal cell carcinomas (RCC) and mutant p53 is a prognostic indicator in RCC ,35. ConsNext, to determine the effect of contact-site mutant R261H on MDCK cell morphogenesis, we generated multiple MDCK cell lines in which R261H mutant was ectopically expressed . The mRNEctopic expression of mutant p53 R163H or R261H cooperates with knockdown of endogenous wild-type p53 to alter cell polarityTo determine the effect of mutant R163H or R261H on the morphological alterations in p53-deficient MDCK cells, we generated multiple MDCK cell lines in which R163H or R261H was ectopically expressed individually along with knockdown of endogenous wild-type p53 -4A. BothLoss of normal morphology is the characteristics of EMT . Given tin vivo epithelial morphogenesis when cultured in a 3-D collagen gel, we developed the 3-D culture model of MDCK cells to study how mutant p53 gain of function is associated with aberrant renal tubulogenesis. Firstly, we found that ectopic expression of mutant p53 R163H or R261H disrupts cyst formation in 3-D culture of MDCK cells. Secondly, we found that mutant p53 R163H or R261H cooperates with p53-KD to disrupt morphogenesis of MDCK cells and form scattered/unordered structures with extensions in 3-D culture. Thirdly, we found that mutant p53 enhances EMT and cooperates with p53-KD to increase the level of c-Met. Taken together, these data indicated that gain of function of mutant p53 alters the normal morphogenesis of MDCK cells via promoting EMT and c-Met expression  is the most common type of kidney cancer and its incidence is increasing . The p53pression . It is noteworthy that tight regulation of cyst size, shape and polarization is critical for normal kidney development and functions. Disruption of these regulatory mechanisms leads to an array of diseases including autosomal dominant polycystic kidney disease, stenosis, and cancer . Our preMutant p53 shows a gain of oncogenic function in driving invasion and metastasis ,44,45. Ic-met proto-oncogene and plays a critical role in epithelial-mesenchymal interaction [c-Met, the receptor for HGF, is the product of the eraction . c-Met reraction ,53. c-Meeraction . It has eraction . Previoueraction . Here, wIn summary, we showed that mutant p53 plays an important role in disrupting tubulogenesis of renal epithelial MDCK cells in 3-D culture and does so through regulating EMT and c-Met. We postulate that ectopic expression of mutant p53 might inhibit p63 activity, or stabilize transcription factors Snail/Slug and Twist, which in turn down-regulates E-cadherin and induces EMT. This process is enhanced by knockdown of endogenous wild-type p53. Moreover, ectopic expression of mutant p53 together with knockdown of endogenous wild-type p53 leads to elevated expression of c-Met, which leads to scattered morphology of MDCK cells in 3-D culture and further promotes cell proliferation and mobility in 2-D culture .Figure S13-D culture of MDCK cells.(TIF)Click here for additional data file.Figure S2Wild-type p53 is not required for tubular formation in MDCK cells.(TIF)Click here for additional data file.Figure S3The schematic representation of p53 siRNA targeting sequence and silent mutations in siRNA-resistant mutant p53 cDNA.(TIF)Click here for additional data file.


The genomic RNAs of influenza A viruses are associated with the viral polymerase subunits  and nucleoprotein (NP), forming ribonucleoprotein complexes (RNPs). Transcription/replication of the viral genome occurs in the nucleus of infected cells. A role for Hsp90 in nuclear import and assembly of newly synthetized RNA-polymerase subunits has been proposed. Here we report that the p23 cochaperone of Hsp90, which plays a major role in glucocorticoid receptor folding and function, associates with influenza virus polymerase. We show that p23 is not essential for viral multiplication in cultured cells but relocalizes to the nucleus in influenza virus-infected cells, which may alter some functions of p23 and Hsp90. Moreover, we show that influenza virus infection inhibits glucocorticoid receptor-mediated gene transactivation, and that this negative effect can occur through a p23-independent pathway. Viral-induced inhibition of the glucocorticoid receptor response might be of significant importance regarding the physiopathology of influenza infections in vivo. The genome of influenza A viruses consists of eight molecules of single-stranded RNA of negative polarity. The viral RNAs (vRNAs) are associated with the nucleoprotein (NP) and with the three subunits of the polymerase complex  to form viral ribonucleoproteins (vRNPs) . An equimolar mix of the amplified products was used as a template for a third PCR reaction, and the resulting amplicon was cloned between the NheI and BstXI sites of plasmid pPolI-PB2-Flag-143 Renilla luciferase in the pPR7-FluA-CAT plasmid The series of eight pPolI plasmids containing the sequences corresponding to the genomic segments of WSN virus, and the four recombinant pcDNA3.1 plasmids for the expression of WSN-PB1, -PB2, -PA and -NP proteins The GR expression vector and GR luciferase reporter plasmid were described previously 5′-GGGGACAACTTTGTACAAAAAAGTTGGCATGCAGCCTGCTTCTGCAAAGTGGTACGATC-3′ and 5′-GGGGACAACTTTGTACAAAAAAGTTGGCAGTTACTCCAGATCTGGCATTTTTTCATCATCAC-3′ for p23, and 5′-GGGGACAACTTTGTACAAAAAAGTTGGCGGAAAGGAAAAGACTCATATCAACATTGTCG-3′ and 5′-GGGACAACTTTGTACAAAAAAGTTGTTAGTTATTTAGCCTTCTGAGCTTTCTGGGCAG-3′ for EF1α). The resulting amplicons were subcloned using the Gateway technology downstream the GST coding sequences into a pCMV-GST plasmid , for construction of the pCMV-GST-p23 and pCMV-GST-EF1α plasmids.The p23 and EF1α coding sequences were amplified from a human spleen cDNA library  with the oligonucleotides All constructs were verified by the sequencing of positive clones using a Big Dye terminator sequencing kit and an automated sequencer (Perkin Elmer). The sequences of the oligonucleotides used for amplification and sequencing can be obtained upon request.−/− MEFs Wild-type and p235 and 3×105 cells, respectively, in a 35-mm dish), using 10 µl of the Fugene 6 transfection reagent (Roche). After 24 hours of incubation at 35°C, the supernatant was removed and replaced with DMEM supplemented with 2% FCS, and the cells were incubated at 35°C for two more days. The efficiency of reverse genetics was evaluated by titrating the supernatant on MDCK cells, in a standard plaque assay using an agarose overlay in complete MEM with 2% FCS. Viral stocks were produced by infecting MDCK cells at a m.o.i. of 0.001 and collecting the supernatant after an incubation of 2 days at 35°C in DMEM supplemented with 2% FCS. Experimental infections were performed at 37°C, unless otherwise indicated.The method used for the production of the WSN-PB2-Strep and P908-WSN-PB2-Strep recombinant influenza viruses by reverse genetics was adapted from previously described procedures 8 cells per 150 mm dishes) with the PB2-wt or PB2-Strep viruses at a m.o.i. of 5 pfu/cell, cells were washed twice with PBS, collected with a cell scraper and centrifuged at 450 g for 5 min. The packed cell volume (PCV) was estimated. Cells were resuspended in 5×PCV of a hypotonic lysis buffer  and kept on ice for 15 mn. NP40 was added at a final concentration of 0.3%, and the lysate was centrifuged at 11,000 g for 2 mn at +4°C. The supernatant corresponding to the cytoplasmic fraction was transferred to a fresh tube, 1∶20 of the volume was frozen at −80°C. The remaining volume was loaded on a 1 ml Strep-tactin column following the recommendations of the supplier (IBA GmbH). Following elution with 6×200 µl of a desthiobiotin solution (IBA GmbH), elution fractions n° 2 to 5 were pooled and concentrated approximately 15-fold using a 10,000 MWCO Vivaspin tube (Sartorius). One third of the resulting sample was subjected to electrophoresis on a 4–15% Tris-Glycine-SDS polyacrylamide gel (Biorad). Following overnight staining of the gel with SYPRO-Ruby (Invitrogen), or western-blotting as described below, the proteins were vizualized using the G-Box (Syngene).At 6 hours following infection of 293T cells .Destaining of SYPRO-Ruby-stained gel slices, reduction, alkylation, trypsin digestion of the proteins followed by peptide extraction were carried out with the Progest Investigator (Genomic Solutions). Peptides were eluted directly using the ProMS Investigator, (Genomic Solutions) onto a 96-well stainless steel MALDI (Matrix Assisted Laser Desorption Ionisation) target plate (Applied Biosystems) with 0.5 µL of CHCA  matrix  were used as internal calibrates. Monoisotopic peak masses were automatically determined within the mass range 800–4000 Da with a signal to noise ratio minimum set to 20. Up to 10 of the most intense ion signals were selected as precursors for Tandem Mass Spectrometry (MS/MS) acquisition excluding common trypsin autolysis peaks and matrix ion signals. In MS/MS positive ion mode, 4000 spectra were averaged, collision energy was 2 kV, collision gas was air and default calibration was set using the Glu1-Fibrino-peptide B ([M+H]+ = 1570.6696) spotted onto fourteen positions of the MALDI target. Combined Peptide Mass Fingerprinting (PMF) and MS/MS queries were performed using the MASCOT search engine 2.1 (Matrix Science Ltd.) embedded into GPS-Explorer Software 3.5  on the NCBInr [20100119 ] database with the following parameter settings: 50 ppm mass accuracy, trypsin cleavage, one missed cleavage allowed, carbamidomethylation set as fixed modification, oxidation of methionines was allowed as variable modification, MS/MS fragment tolerance was set to 0.3 Da. Protein hits with MASCOT Protein score ≥83 and a GPS Explorer Protein confidence index ≥95% were used for further manual validation.Raw data for protein identification were obtained on the 4800 Proteomics Analyzer (Applied Biosystems) and analyzed by GPS Explorer 2.0 software (Applied Biosystems/MDS SCIEX). For positive-ion reflector mode spectra 3000 laser shots were averaged. For MS calibration, autolysis peaks of trypsin ([M+H]5 cells in a 35-mm dish) using 10 µl of the Fugene-6 transfection reagent (Roche). At 48 hours post-transfection, cells were lysed in 300 mL of Tris-HCl [pH 7.4] 50 mM, NaCl 120 mM, EDTA 1 mM, NP40 1% and Protease Inhibitor Cocktail 1X (Sigma). Gluthation Sepharose 4 Fast Flow beads  were added to the supernatant and incubated overnight under gentle rotation. Beads were washed three times in Tris-HCl [pH 7.4] 50 mM, NaCl 120 mM, EDTA 1 mM, NP40 1%, and bound proteins were eluted by incubation with 30 µl of Laemmli buffer.Plasmids pCMV-GST-p23, pCMV-GST-EF1α or pCMV-GST (1 µg) were transfected together with pcDNA3.1-PB1, -PB2, and/or -PA (1 µg) into a subconfluent monolayer of 293T cells (8×105 cells per well in 24-well-plates) were transfected with anti-p23 or control siRNAs  at a final concentration of 25 nM using the DharmaFECT reagent (Dharmacon) according to the manufacturer's recommendations. For viral minigenome assays, plasmids pcDNA3.1-PB1, -PB2, -PA and -NP  were transfected together with the pPR7-FluA-Luc plasmid (0.1 µg) using 5 µl of the Fugene-HD transfection reagent (Roche). At 24 hours post-transfection, cell lysates were prepared and luciferase activity was measured, using the Lysis Buffer and substrate provided in the Renilla Luciferase Assay System kit (Promega) and a Tecan luminometer (Berthold).Subconfluent monolayers of 293T cells (2×105 cells per well in 12-well-plates) were pre-incubated for 16 hours in serum free medium prior to cotransfection with a GR expression vector, a GR-Firefly luciferase reporter plasmid and a pTK-Renilla luciferase expressing vector (0.5 µg of each plasmid) using 5 µL of the FuGENE HD reagent (Roche). At 24 hours post-transfection cells were infected at a m.o.i. of 5 pfu/cell with the A/WSN/33 virus. After one hour of adsorption, the viral suspension was replaced with medium supplemented with dexamethasone and cells were incubated at 35°C for 10 hours. Cell lysates were prepared and luciferase activities were measured using Luciferase Assay kits (Promega).For GR-mediated gene transactivation assays, subconfluent monolayers of 293T cells (4×10293T cells on coverslips were transfected using the FuGENE HD reagent (Roche) with a PB2-flag expression vector alone or in combination with PB1 and PA expression vectors. At 24 h post-transfection cells were fixed with PBS-4% paraformaldehyde for 20 min and permeabilized with PBS-0.1% Triton X100 for 15 min. They were incubated with a mixture of the mouse monoclonal anti-p23  and the rabbit polyclonal anti-Flag  antibodies, and then with a mixture of AF555-coupled anti-mouse IgG and AF488 coupled anti-rabbit IgG secondary antibodies .A549 or 293T cells on coverslips were infected with the PB2-HA recombinant virus at a m.o.i. of 5 pfu/cell or mock-infected, and incubated at 37°C. At 4–8 hours post-infection, cells were fixed and permeabilized as indicated above. They were incubated with a mixture of the mouse monoclonal anti-p23 and the rabbit polyclonal anti-Hsp90  antibodies, and then with a mixture of AF640-coupled anti-mouse IgG secondary antibody , AF555-coupled anti-rabbit IgG secondary antibody , and AF488-coupled anti-HA antibody . Alternatively, cells were incubated with dexamethasone 10 nM for 1 hour at 37°C prior to fixation and permeabilization. They were incubated with a mixture of the rat anti-HA  and mouse anti-glucocorticoid receptor  monoclonal antibodies, and then with a mixture of AF555-coupled anti-mouse IgG secondary antibody  and AF488-coupled anti-rat IgG secondary antibody . The samples were analyzed under a fluorescence microscope (Zeiss Axioplan 2 imaging- Zeiss ApoTome).For western blot assays, cells in 35-mm dishes were resuspended directly in 300 µl of sample loading buffer. The cell lysates were centrifuged for 2 mn at 16,000 g on a QIAShredder column (QIAGEN), heated for 3 min at 95°C and analyzed by electrophoresis on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and western blotting using PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies directed against p23 , GST , Hsp90  or Histone3 , or with rabbit polyclonal serum directed against the PB1, PB2, or PA proteins  or against A/PR/8/34 virions  influenza A virus expressing a PB2 protein fused to a Strep-tag epitope at the C-terminus (PB2-Strep virus). The Strep-tag is a short polypeptide which binds specifically to Strep-tactin, a derivative of streptavidin 9 293T cells at a multiplicity of infection (m.o.i.) of 5 pfu/cell. Cytoplasmic extracts were prepared at 6 hours post-infection (hpi), and were loaded onto Strep-tactin columns. Native PB2-Strep complexes were eluted using desthiobiotin, and subjected to SDS-PAGE analysis. As shown in The PB2-Strep and wild-type (PB2-wt) viruses were used in parallel to infect 4×10Co-purification assays were then performed using a GST-p23 fusion protein transiently expressed in 293T cells. A pCMV-GST-p23 expression vector was transfected in 293T cells together with pcDNA3.1 expression vectors for the WSN-PB1, -PB2 or -PA proteins, separately or in combination. Similar to p23, EF1-α is an abundant, constitutively expressed, cytoplasmic protein, and therefore a GST-EF1α construct was used in parallel as a control. At 48 h post-transfection, total cell extracts were prepared and incubated with glutathion beads as described under the −/−) or from control wild-type mice (wt) −3 pfu/cell, viral titers in the culture supernatants were determined at different times post-infection by plaque assay on MDCK cells. The WSN virus replicated at a similar rate on p23−/− and wt cells, titers of 107–108 pfu/ml being observed at 48 hpi .To further document the effect of p23 depletion on influenza virus replication, we performed single-cycle growth assays on p23/− cells . The virSingle-cycle growth assays were also performed in 293T cells transiently transfected with anti-p23 siRNAs. The steady-state level of p23, as evaluated by western-blot analysis of serial dilutions of total cell extracts, showed a significant reduction in 293T transfected with the anti-p23 siRNAs as compared to the control siRNAs .The subcellular localization of p23 in influenza virus- and mock-infected A549 and 293T cells was compared. A recombinant WSN virus expressing a PB2 protein fused with the HA tag at the C-terminus was used in these experiments, to allow simultaneous labeling of the viral PB2 and cellular p23 and Hsp90 proteins. The anti-HA antibody did not recognize the WSN hemagglutinin, as expected from the fact that the YPYDVPDY sequence of the HA tag is not present in this hemagglutinin, and as demonstrated by the background levels of fluorescence measured on control cells infected with the wild-type WSN virus . In uninThe subcellular localization of p23 was then examined in transfected cells transiently expressing the viral polymerase subunits. Relocalisation of p23 to the nucleus was clearly observed in A549 or 293T cells transiently expressing the PB2 subunit, either alone or in combination with the PB1 and PA subunits . Overallvs mock-infected cells, 293T cells were cotransfected with a GR expressing plasmid, a reporter plasmid containing the Firefly-luciferase gene under the control of a minimal TK promoter and GR-responsive elements, and a Renilla-luciferase expressing construct. At 48 hours post-transfection, they were infected with WSN at a m.o.i. of 5 pfu/ml, or mock-infected. After one hour of adsorption, the viral inoculum was removed, and replaced with fresh medium either supplemented with dexamethasone or not. After 10 hours of stimulation, cell lysates were prepared and Firefly-luciferase activities were determined and normalized with respect to Renilla-luciferase activities. As shown in Firefly-luciferase gene in infected cells  was reduced as compared to in mock-infected cells . The subcellular localisation of the GR was examined in WSN-PB2-HA- and in mock-infected 293T cells, upon incubation in the absence or in the presence of 10 nM dexamethasone. An indirect immunoassay was performed at 5 hpi, using a mixture of an anti-HA and an anti-GR antibody. Uninfected cells generally showed cytoplasmic or cytoplasmic and nuclear GR staining in the absence of dexamethasone   also contained the Hsp90 chaperone, in agreement with existing data showing PB2-Hsp90 and p23-Hsp90 interactions Whereas Hsp90 is thought to play a role in the import and assembly of newly synthetized influenza polymerase subunits The usually observed nucleo-cytoplasmic shuttling of p23 is related to its complex role in glucocorticoid signalling. In the absence of hormone, glucocorticoid receptors (GRs) are mainly located in the cytoplasm, and p23 promotes their folding into a hormone-binding conformation by stabilizing GR-Hsp90-p60-Hsp70 complexes This complex regulatory function of p23 at various steps of the GR signalling pathway is likely to be disrupted by the strong relocalisation of p23 in the nucleus of influenza-virus infected cells. Indeed, we found that the GR sub-cellular localisation is altered and the GR-mediated gene transactivation is impaired in influenza virus-infected cells. Viral infection still shows a negative effect on GR-mediated gene transactivation in cells that are defective for p23. This observation, although it does not rule out a contribution of p23, indicates that viral-induced inhibition of GR-mediated signalling can occur through a p23-independent and redundant pathway. A possible approach to unravel the contribution of p23 would be to identify influenza variants defective for p23 binding and to compare their impact on GR signalling.Our observation of an impaired response to glucocorticoids in influenza infected cells adds to few previously published data showing alterations of the GR activity upon viral infections  were infected at a m.o.i. of 10 pfu/cell with the WSN-PB2-HA virus. At 5 hpi, cells were fixed, permeabilized and stained with antibodies specific for the HA tag (PB2) and for the Hsp90 protein. Samples were analyzed under a fluorescence microscope (Zeiss Axioplan 2 Imaging - Zeiss ApoTome). A merge of the signals corresponding to DAPI (blue), HA (red) and Hsp90 (green) is shown.(TIF)Click here for additional data file.Figure S2Specific recognition of the HA-tag and not the WSN virus hemagglutinin by the anti-HA antibody. A549 (A) or 293T (B) cells were infected at a m.o.i. of 10 pfu/cell with the WSN-PB2-HA virus (upper panels) or the WSN wild-type virus (lower panels). At 8 hpi (A) or 6 hpi (B), cells were fixed, permeabilized, and stained with antibodies specific for the HA tag (PB2) and for the p23 protein. Samples were analyzed under a fluorescence microscope (Zeiss Axioplan 2 Imaging - Zeiss ApoTome). A merge of the signals corresponding to DAPI (blue), HA (green) and p23 (red) is shown.(TIF)Click here for additional data file.Figure S3Subcellular localisation of p23 in 293T cells transiently expressing the viral PB2 or NP protein. 293T cells were transfected with a plasmid encoding NP (middle pannel), PB2-Flag (lower panel) or mock-transfected (upper panel). At 24 hpi cells were fixed, permeabilized, and stained with antibodies specific for the Flag tag (PB2) or the NP protein, together with an anti-p23 antibody. Samples were analyzed under a fluorescence microscope (Zeiss Axioplan 2 Imaging - Zeiss ApoTome). A merge of the signals corresponding to DAPI (blue), Flag or NP (green) and p23 (red) is shown.(TIF)Click here for additional data file.