However, passive [Ca2+]i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca2+]i changes is difficult to attain. The tools designed so far to modify [Ca2+]i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electro-optical technique to quantitatively set [Ca2+]i, in real time and with sub-cellular resolution, using two-photon Ca2+ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca2+]i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca2+ channels, and the standardization of [Ca2+]i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling.The variations of the intracellular concentration of calcium ion ([Ca Finally, in the third technique, single-photon ultraviolet pulses trigger the photolysis of a Ca2+chelator. Throughout the 90′s, Neher and his group intensively used this to investigate the cellular mechanisms involving Ca2+ at the synapse 2+]i peaks and decays, which differ from the continuous variations experienced by cells under physiological conditions. Moreover, the excitation volume defined by the UV flash is spatially extended, making it difficult to perform quantitative Ca2+ release with high spatial resolution.While [Ca2+]i with sufficient temporal and spatial resolution to mimic naturally occurring intracellular Ca2+ signals. We demonstrate the capabilities of this tool by mimicking voltage-dependent Ca2+channels in neurons. The theory of our technique and its experimental design are discussed. We focus on three applications, namely the independent control of the membrane potential and [Ca2+]i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca2+ channels, and the standardization of [Ca2+]i patterns across different cells.The present study describes a new technique that solves many of these problems. Specifically, we have developed a tool to control i variations, we monitored these with fluorescence imaging using fluo-4 as the Ca2+ indicator and a second laser . We then played back the membrane potential recordings to control Ca2+ uncaging. Finally, the cross-correlation between the endogenous and the photolytically-induced variations of ΔF/F was evaluated at zero time lag. A cross-correlation of 100% means both signals have exactly the same dynamics, whereas 0% means they are independent.To verify that DTC can simulate endogenous i and membrane potential variations. To date, only Ca2+channels blockers have been effective at decoupling these by blocking the Ca2+ influx altogether for a particular sub-population of channels, effectively preventing the Ca2+ influx from depending on membrane potential 2+]i while the membrane potential is held constant. To begin, the membrane potential (V0(t)) and fluorescence variations (F0(t)) are recorded when the neuron is spiking (2). These recordings serve as a reference. The neuron is then hyperpolarized below the activation threshold of L-type Ca2+ channels, causing these endogenous channels to close and the Ca2+-associated fluorescence signal to flatline . As expected, F0 and F1 are not significantly correlated and XC0,1(0) is down to 8%±15% (n = 56). When DTC is activated (3), the uncaging restores the [Ca2+]i variations with dynamics significantly correlated to the endogenous ones: XC0,1(0) = 88%±8% (n = 56). The cross-correlations with and without uncaging are also verified to be significantly different from each other (p<0.001). Thus, DTC effectively simulates the endogenous L-type Ca2+ channels even though the membrane potential is held below threshold.First, we demonstrate that DTC can effectively dissociate [Ca spiking  and 2A2ctivated  and 2A32+ channels are blocked pharmacologically and DTC is used to functionally reinstate the [Ca2+]i variations. To do this, we first record a reference, as before  = 32%±20%, n = 17). With DTC, the [Ca2+]i variations are restored to their original level  = 86%±13%, n = 17, significantly higher than playback without uncaging, p<0.001). That is, DTC effectively re-introduces “virtual” L-type Ca2+ channels in the soma to replace those that were pharmacologically blocked. Interestingly, uncaging while the Ca2+ channels are blocked leads to similar results as uncaging under hyperpolarization but without blockers (i.e. experiment 1), as shown by the high cross-correlation for this subset  = 91%±4%, n = 17, p = 0.243).In a second application, the endogenous Cas before . Then, 42+]i variations between different cells. The procedure is the same as for application 1 (0) are played back in a different neuron (neuron1) that is hyperpolarized. Because the cytoplasms of both neurons are dialyzed by the content of the patch pipette, the Ca2+ buffers inside both neurons are also quantitatively and qualitatively similar: the playback of the membrane potential of neuron0 thus induces the same [Ca2+]i variations in both neuron0 and neuron1  = 93%±6%, n = 108).In a third application, DTC is used to standardize the i variations, the same model parameters were used to reproduce up to four different sequences in a given neuron. The measured [Ca2+]i variations all reached similar levels  = 91%±5% n = 26, 89%±6% n = 26, 87%±9% n = 13 and 91%±3% n = 2, Friedman test, p = 0.194). Reproducibility was further confirmed with back to back sequences, differences being on average 0%±2%, n = 401, p = 0.491 (see Methods).Finally, to verify the robustness of the L-type Ca2+]i with high enough spatio-temporal resolution to simulate the cellular variations of [Ca2+]i caused by endogenous channels. It relies on the regulated release of Ca2+ by uncaging with two-photon laser, whose power is modulated by a dynamic-clamp plug-in. To our knowledge, such precise control of [Ca2+]i dynamics has never been achieved before. DTC therefore provides a new and versatile tool for the study of cellular function, with capabilities that include the dissociation of membrane potential from [Ca2+]i variations and the standardization of [Ca2+]i patterns through the virtual knock-in of calcium-specific, user-defined conductances.In summary, DTC provides a dynamic control of [Ca2+ release volume 2+]i variations, and their spatial location. Whereas blockers act indiscriminately in both the soma and dendrites, DTC can be applied more selectively by targeting the laser focus to either or both, enabling the user to gauge the effect of subcellular location of Ca2+ influx. Thus, DTC provides the tool to test hypotheses related to key properties of Ca2+ signaling, such as the functional importance of subcellular domains, or the impact of slowing or speeding the rate of Ca2+ influx.DTC is based on two-photon uncaging, which has several advantages: two-photon uncaging both defines a 3D spatially confined Ca2+ over Mg2+ (KdCa 80 nM vs. KdMg 9 mM), unlike DM-nitrophen (5 nM vs. 25 µM). Therefore, with physiological concentrations of Mg2+ in the pipette  and a lower affinity when photolyzed (3 vs. 1 mM), and it exhibits a higher two-photon cross-section (0.01 vs. 0.001 GM). The lower affinity allows DM-nitrophen to be loaded above 95% for [Ca2+]i ≈100 nM, while under the same conditions (same [Ca2+]i and same amount of free Ca2+ buffer), NP-EGTA could only be loaded slightly above 50% . Moreover, NP-EGTA would be restricted to very low concentration (around 200 µM) to allow signal monitoring with 100 µM fluo-4. The combined effect of low concentration of caged NP-EGTA and lower two-photon cross-section means that NP-EGTA would require more than 10 times more optical power (536 mW instead of 47 mW to attain 1000 pA Ca2+ current), which would be difficult to achieve with our current setup, in addition to being potentially detrimental to the preparation. A possible solution is to increase both NP-EGTA and fluo-4 concentrations, though this may be ill-advised for two reasons. First, the NP-EGTA would still be only half loaded, which may cause the Ca2+ to be released only transiently rather than continuously, as freshly released Ca2+ is likely to be quickly recaptured by non-photolyzed neighboring empty cages 2+ buffer, which might distort the endogenous Ca2+ signal 2+ buffers (free NP-EGTA + free fluo-4). Thus, we conclude that whenever Mg2+ is not crucial for the study in question (the case here), then DM-nitrophen seems to be a better choice than NP-EGTA for the application of DTC.While several cages have been reported in the literature 2+. As noted above, only 47 mW of laser power is required to attain a 1000 pA Ca2+-current when using DM-nitrophen, while a typical current in mitral cells is usually less than 150 pA 2+ pool in a static release volume, i.e. if the laser were kept parked at one spot. Because the laser is instead scanned along the inner membrane surface, the swept release volume is about 90 times larger than the focal volume (about 40 fL for a 15 µm diameter cell). Therefore, the local depletion is about 0.1% and can be readily replenished by diffusion from non-illuminated neighboring regions, as experimentally verified when comparing back to back sequences.Local depletion is yet another risk to be aware of when uncaging Ca2+ reporter is fluo-4. This was chosen because it provides several advantages compared to other available Ca2+ reporters. First, with its absorption peak at 494 nm d (345 nM) makes fluo-4 one of the best choices to monitor both single elementary events  and larger trends in Ca2+ variations As noted above, our Ca2+ spark or sparklet from a single channel 2+ influx. Nevertheless, when considering the Ca2+ influx produced by a local population of channels (the channels distributed in the plasma membrane of the soma in our case), their averaged signals over the time scale of interest can be faithfully reproduced, as experimentally demonstrated here. Working with a population of channels allows us to use the deterministic Hodgkin-Huxley formalism to model this global conductance. If needed, a random component can also be introduced to mimic population stochastic response Since DTC uncaging is light-based, its spatial resolution cannot be better than the diffraction limit. The water-immersion objective used here has a numerical aperture of 0.95, which is close to the maximum commercially available while remaining compatible with patch clamp (i.e. with a working distance over 2 mm). With our setup see , the relHow fast can our DTC be pushed? In this set of experiments, we use a refresh rate of 10 kHz for the dynamic-clamp. This is about one order of magnitude faster than the fastest time constant of the conductance we mimic cf.  and is t2+]i at a set value. To mimic voltage-dependent Ca2+ channels, DTC does not rely on feedback, and instead is based only on a feed-forward mechanism, as illustrated in 2+]i is never used to compute how much Ca2+ is to be released. Second, a proper clamp would involve a way to actively modulate the Ca2+ concentration both upward and downward. In our case, DTC only achieves the upward modulation: the downward modulation is ensured by the cell Ca2+ clearance mechanisms, as is the case when Ca2+ enters the cell through voltage dependent Ca2+ channels. While in principle it may be possible to introduce an active Ca2+ scavenger in addition to our Ca2+ chelator, the only photoactivatable scavenger commercially available is, to our knowledge, Diazo-2  and DTC can be programmed to mimic this as well, provided an appropriate CICR model is implemented. In this case, the RTXI input would not be the membrane potential but rather a measure of the actual [Ca2+]i , for example obtained by fluorescence monitoring with a Ca2+-sensor. With the appropriate Ca2+ cage, such an implementation of DTC would be all-optical and allow simultaneous multicellular control. Finally, DTC need not be restricted to caged Ca2+. For example, caged neurotransmitters can be used to reproduce localized synaptic release with controlled dynamics. Alternatively, caged-second messengers such as inositol trisphosphate can be used to study second messenger dynamics in sub-cellular compartments.It should be noted that DTC can, in principle, be applied to quantitatively control the release of any caged compound with adequate two-photon cross-section. For example, another mechanism through which Ca2+ in neurons, where we show that the combination of a dynamic-clamp with two-photon uncaging enables the release of caged compounds with an unprecedented degree of control and spatiotemporal resolution. This technique is robust and easy to implement with standard two-photon microscopes, making it attractive as a general tool for the study chemical signaling in cells.In short, DTC is a general technique with many potential ramifications. Our goal in this study is to lay the groundwork for this technique, with the hope of establishing it as a new tool for the study of cellular dynamics. Specifically, we have confined our proof of concept to the modulation of CaLong-Evans rats . Briefly, after a deep, isofluorane-induced anesthesia, confirmed by absence of reaction to toe pinching, the animal was decapitated and the head submerged in ice-cold sucrose aCSF (see composition below). The olfactory bulbs were removed, sliced with a Vibratome 1500 , and placed in regular aCSF bubbled with 95% O2-5% CO2 to attain a pH of 7.3–7.4. Slices were kept at room temperature until individually transferred into the recording chamber where experiments were carried out. One neuron was patched per slice, with a borosilicate glass pipette pulled on a Sutter P97 puller from BF120-69-10 capillaries (3–6 MΩ).The following protocol was approved by the Boston University Institutional Animal Care and Use Committee (IACUC Protocols # 07–014 and 10–021) and everything was done to minimize the number of animals and their suffering. We prepared parasagittal olfactory bulb slices (350 µm) from weaned sub-adult 3, 1 NaH2PO4, 2.5 KCl, 10 glucose, 2 MgCl2, 1 CaCl2. The sucrose aCSF used for the dissection and slicing is the same, but with the 120 mM NaCl replaced by 240 mM sucrose and the MgCl2 increased to 3 mM to compensate for the removal of CaCl2. Both solutions have the same osmolality  and pH (7.3–7.4). L-type calcium channels 2ATP, 0.47 NaxGTP, 1.56 CaCl2, 1.585 DM-nitrophen and 0.1 K5fluo-4. Therefore, the concentrations of loaded DM-nitrophen, free Ca2+, and free fluo-4 are, respectively 1.51 mM, 104 nM and 77 µM.Both the room temperature incubation and subsequent recording at 31°C used artificial cerebro-spinal fluid (aCSF) composed of (in mM): 120 NaCl, 25 NaHCOAll chemicals were obtained from Sigma-Aldrich, with the exception of DM-nitrophen and fluo-4, obtained from Invitrogen.2+ fluorescence traces could be compared between runs and conditions, the membrane potential variations had to be kept identical between different traces. Therefore, our custom-made stimulation and recording program (Matlab and Labview) included a playback mode. This mode outputs previously-recorded sequences of membrane potential variations through a National Instruments card (PCI-6221 with a BNC-2110) and sends them to either the amplifier or dynamic-clamp setup . If µ0 and σ0 are respectively the mean and standard deviation of F0 (similarly for F1), then XC0,1 (τ) is given byTo compare the dynamics of two fluorescent traces of N points (F0 and F1), we evaluated their cross-correlation Furthermore, two traces are significantly correlated with a 5% threshold Unless stated otherwise, we used only significantly correlated traces. To test for reproducibility between sequences, some sequences (n = 401) were repeated back to back, separated by a 1s interval. Since the differences were not statistically significant (p = 0.49), only the highest of the two cross-correlation coefficients was kept for subsequent analysis.www.rtxi.org), connected through a National Instruments card PCI-6040E and a plug-in with a model of the L-type Ca2+ conductance. This samples the membrane potential V(t) and calculates the value of the conductance every 100 µs (10 kHz refresh rate). The corresponding current, namely ICa(V(t)), is then computed based on V(t) and a locally applied Ohm's law. Since an ionic current corresponds to a number of charges flowing through a surface per unit of time, we thus determine the number of calcium ions (ΔNCa) required to produce such a current, for a given time step.Experiments were performed using the Real-Time eXperiment Interface  is set to be the sampling period of the voltage by the dynamic-clamp software, which is much faster than typical time constants involved in the dynamics of the Ca2+conductances (see The time step (Δtnces see . The sur2+ ions:The above provides us with the first relationship between the current and the number of Cae is the elementary charge and V(t) the membrane potential.where cL far below the onset of pulse saturation 2+ions ) is therefore linearly proportional to the actual caged Ca2+available ([CCa2+]0), at any point in space  The laser in our system is operated at an intensity Iwhere α, the photolysis rate, is defined as2 is the two-photon photolysis cross-section and we have adopted a 3D Gaussian model for the focal volume, with lateral and axial waists rw and zw respectively. We further adopt the notation2+]0 is roughly a constant, i.e. not depleted by the uncaging process itself. This assumption is justified in the discussion and experimentally confirmed.where σCa,uncaging and ΔNCa,conductance during the interval ΔtDC. The total number of Ca2+ ions (ΔNCa) liberated in the release volume (Vr) can be expressed asThe principle of DTC is to match ΔNA, Avogadro's number, converts the result of the volume integral from moles into number of ions.where NCa, the current calculated by the dynamic-clamp, we then control the intensity of the laser beam to reproduce the Ca2+ variations in the cell in real time. The mean of the squared intensity Since f is the laser repetition rate, pt is the pulse duration and gp(2) is the second-order temporal coherence factor.where An electro-optic modulator (EOM) controls the laser power, which, for wavelength λ, is given byCa(V(t)) (see conductance model below), the rest being just constant scaling factors.since the area of the beam at the focal plane is max) is adjusted to provide a linear control of the optical power (0 to Pmax). The voltage output of RTXI then becomesFinally, the input voltage range of the EOM ) being the activation gate, i(V(t)) the inactivation gate, and gSee 0,1(0) between conditions were statistically significant, we used the Wilcoxon signed rank test, unless stated otherwise. This non-parametric test was chosen since some sets were not normally distributed (as tested with a Shapiro-Wilk test). However, the medians and means were close enough to report the results as mean ± S.D as usual.To determine whether the differences observed in XC


Recent advances in DNA sequencing have revealed that AML-NK is molecularly heterogeneous with >30 genes recurrently targeted by somatic mutations in this disease.2 What is also evident is that each individual case of AML-NK appears to harbor only a small number of coding driver mutations, often as few as three and usually no more than five.3 Furthermore, it is manifest that the precise combination of driver mutations in the genome of each AML impacts on its salient features, including responsiveness to treatments and prognosis.3Acute myeloid leukemia (AML) is the commonest myeloid malignancy, yet there has been little therapeutic progress for this disease in decades, and only 25–30% of patients survive long term.5 Here, we report that the two most commonly co-occurring somatic mutations in AML, namely Nucleophosmin (NPM1) exon 12 mutations (NPM1c) and internal tandem duplications of FLT3 (FLT3-ITD), cooperate explosively to induce AML in knock-in mice. In revealing this striking molecular synergy, our work offers a basis for the frequent co-occurrence of these two mutations and provides a valuable model for in-depth studies of the pathogenesis and treatment of this large subgroup of AML.These observations provide a sound starting point for systematic mechanistic studies to understand the pathogenesis and improve the treatment of AML-NK. Carefully designed mouse models are the gold standard in the study of normal and malignant hemopoiesis, and are already instructing our understanding of AML-NK.6 This ability is impaired in 30% of AMLs as a result of NPM1c mutations, which disrupt the nucleolar localization signal of NPM1 and generate a nuclear export signal in its place.7 Mutant NPM1 is known to bind to and alter the subcellular distribution of several proteins, including HEXIM1, p19Arf and nuclear factor-κB;8 however, the relevance of these interactions to AML is unclear. FLT3-ITD mutations occur in 20–25% of AML9 and result in ligand-independent receptor dimerization and constitutive FLT3 signaling,10 and are associated with an increased risk of relapse. Moreover, patients with low or absent levels of wild-type (WT) FLT3, consistent with loss-of-heterozygosity (LOH) for this locus, have a particularly poor outcome.9NPM1 is a nucleolar phosphoprotein involved in many cellular processes. For many of its roles, it relies on its ability to shuttle between the nucleolus, nucleus and cytoplasm using subcellular localization signals.Npm1c mice, in association with specific recurrent mutations including activating insertions in Csf2 and Flt3.4Flt3-ITD homozygous mutant mice exhibit enhanced proliferation and survival properties in hemopoietic progenitors and develop a late-onset disease akin to chronic myelomonocytic leukemia.11Recently, we described a conditional knock-in mouse model of NPM1c mutations and demonstrated that one-third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We went on to show that insertional mutagenesis with transposons led rapidly to AML in 80% of NPM1c with FLT3-ITD we crossed conditional Npm1flox−cA/+ with constitutive Flt3ITD/+ to generate Npm1flox−cA/+; Flt3ITD/+ double heterozygous mice, then crossed into Mx1-Cre transgenic mice to induce recombination of Npm1flox−cA in hematopoietic stem cells.4 The Mx1-Cre allele requires induction by interferon, usually achieved by intraperitoneal injection of polyinosinic-polycytidylic acid(pIpC). However, we observed universal and rapid emergence of AML (myeloid leukemia with maturation) in uninjected Npm1flox−cA;FLT3ITD/+;Mx1-Cre+ mice (hereafter referred to as ‘Npm1c/Flt3-ITD mice'). Mx1-Cre is known to ‘leak' in 2–4% of hemopoietic stem/progenitor cells,12 and this was sufficient to rapidly generate AML from double mutant cells signifying a striking cooperativity between Npm1c and Flt3-ITD. The presence of the cytoplasmic NPM1 was confirmed on protein blots . By contrast, no case of AML was observed in Npm1flox−cA;Mx1-Cre+ mice , FLT3ITD/+ mice (hereafter referred to as ‘Flt3-ITD mice' n=34) or WT mice  aged to at least 8 months  and CD45dim , Flt3-ITD (n=4), WT (n=4) and Npm1c/Flt3-ITD (n=4) were studied in serial replating assays. Npm1c/Flt3-ITD cells gave rise to significantly more colonies at first and subsequent platings than any other genotype  and hepatomegaly  at the time of death. Npm1c/Flt3-ITD leukemias were transplantable into both syngeneic and NOD SCIDγ mice demonstrating their true neoplastic nature (data not shown).Blood smears from sick mice confirmed the presence of blasts, and histological sections demonstrated widespread infiltration of solid organs by abnormal myeloid cells . Cells iNpm1c and Flt3-ITD, the two most commonly co-occurring AML mutations, is rapidly and universally leukemogenic in knock-in mice. These findings are particularly striking in light of the fact that, in isolation, both Npm1c4 and Flt3-ITD11 mutations have relatively subtle effects on mouse hemopoiesis and lead to leukemia or a myeloproliferative disorder only after prolonged latencies and in a minority of mice.AML is a molecularly and clinically heterogeneous disease and recent studies have revealed that this heterogeneity is derived, to a large extent, from the specific combinations of somatic driver mutations present in individual cases. Here, we show that the combination of Npm1c/Flt3-ITD mice, which suggests either: (i) that the two mutations are sufficient to promote AML in this strain of mice (C57BL6/N) or (ii) that additional mutations are acquired very rapidly in the pool of cells susceptible to leukemic transformation. The later possibility is supported by the fact that at least one type of somatic mutation, namely LOH for Flt3-ITD, was frequently observed in our mouse AMLs over this short time span. We recently reported that Npm1c can generate AML in collaboration with, amongst others, activating insertions of the GrOnc transposon in intron 9 of mouse Flt3. These insertions led to aberrant expression of a Flt3 messenger RNA predicted to code for an amino-terminal truncated version of Flt34 which, like Flt3-ITD, was thought to be constitutively active. Most of these murine AMLs harbored additional transposon insertions thought to be important in leukemogenesis. Thus, at this stage it appears more likely that additional mutations may be required for leukemogenesis in our Npm1c/Flt3-ITD mice, but this cannot be stated unequivocally.What is most remarkable about our findings is the very short latency of AML in Flt3-ITD with NUP98-HOXD13 in mice led to AML after a much longer latency (median 95 days),14 despite the fact that, unlike Npm1c, NUP98-HOXD13 alone leads to a highly penetrant myelodysplastic syndrome with a high risk of leukemic transformation. This relative delay is particularly intriguing as NUP98-HOXD13 can promote leukemic transformation in association with simple overexpression of WT FLT3.15 By contrast, in two large transposon-mediated insertional mutagenesis screens, one published4 and one ongoing, we never observed transposon insertions leading to simple Flt3 overexpression amongst >100 mouse Npm1c +ve AMLs.In interesting contrast to our present work, a recent report demonstrated that the combination of Npm1c and Flt3-ITD. In the context of a stochastic model for AML pathogenesis,2 this potent molecular synergy goes some way toward explaining why NPM1c and FLT3-ITD co-occur so frequently and make the model described here a valuable tool for the study of the pathogenesis and treatment of one of the largest molecularly defined subgroups of AML.Notwithstanding the above, our observations emphasize the remarkable complementarity between


Haem oxygenase-1 (HO-1) is increasingly regarded as a pro-tumoral target in the treatment of human cancers. Currently, little is known about HO-1 and its role in human acute myeloid leukaemia (AML) to regulate apoptosis in response to chemotherapy. Recently, we showed that HO-1 protects AML samples from tumour necrosis factor-α (TNF)-induced apoptosis - it being regulated by transcription factors Nrf2, NF-κB and AP-1. This study aims to analyse the role of HO-1 in regulating apoptosis in AML cells in response to two front-line chemotherapeutic agents used for AML, cytarabine and daunorubicin. Here we show that HO-1 expression in AML samples was increased in response to both cytarabine and daunorubicin treatment, and micro RNA (miRNA) silenced HO-1 expression in combination with either daunorubicin or cytarabine induced a greater apoptotic responses in AML cells. Moreover, we showed that both daunorubicin and cytarabine induced reactive oxygen species (ROS) accumulation to induce apoptosis in AML. However, ROS-dependent induction of HO-1 was limiting the apoptotic response that is seen in AML towards cytarabine and daunorubicin treatment. These findings suggest concurrent inhibition of HO-1 expression in conjunction with chemotherapeutic treatment would improve the number of cases who reach complete remission. Acute myeloid leukaemia (AML) is a malignancy of haemopoietic progenitor cells  and accoA number of mechanisms have been suggested to contribute to drug-resistance in AML. These include, the targeted cells fail to undergo apoptosis in response to the chemotherapy agent, drugs failing to reach their intracellular targets or the removal by the ABC membrane transporter protein, P-glycoprotein (Pgp). Pgp is a efflux transporter, present within the cell's plasma membrane, and its expression in AML has been reported to be relatively low, however an increase in its expression after drug treatment and also at the point of relapse, have been reported . MoreoveRecently, we reported that haem oxygenase-1 (HO-1) has an important function in protecting human AML cells from TNF-induced apoptosis . To dateFurthermore we have recently we showed that HO-1 is in fact down-regulated in AML by their constitutively active NF-κB activity present, and that inhibiting NF-κB brings HO-1 levels back to more normal levels, providing further secondary protection for AML cells against NF-κB inhibition. Here were undertaken to investigate the role of HO-1 in regulating cytoprotective responses to two common front-line chemotherapy agents, cytarabine and daunorubicin, which are currently widely used in treating patients presenting with AML.To understand the mechanisms of chemoresistance in AML cells to cytarabine and daunorubicin, we have examined the levels of apoptosis of primary AML samples and AML cell lines in response to varying concentrations of these drugs. The drug concentrations selected were based on previous studies , 27. OneSince we have previously shown that HO-1 protects AML cells from apoptotic stimuli, we wanted to determine if either cytarabine or daunorubicin could induce the expression of this cytoprotective gene in AML cells. Figure We developed a lentivirus-based miRNA delivery system on a GFP backbone to target HO-1 mRNA knockdown and silence HO-1 expression in AML cells. Figure 2DCFDA oxidation occurs in response to the proteasome inhibitor bortezomib and also in response to NF-κB inhibitor BAY 11-7082 [2DCFDA oxidation in U937 and THP-1 cells in response to cytarabine and daunorubicin treatment over a 6 h time period. N-acetylcysteine (NAC) is an antioxidant scavenger which quenches ROS activity present within the cellular environment. NAC was used to determine if removing ROS would block cytarabine- and daunorubicin-induced HO-1 expression. Finally, we wanted to determine whether NAC could block apoptosis induced by cytarabine and daunorubicin. Figure We have previously shown in AML cells that H 11-7082 , 30. We In this study we have investigated the role of HO-1 in protecting AML cells from apoptosis in response to cytarabine and daunorubicin. We have previously shown that HO-1 can protect AML cells from apoptosis in response to the proteosome inhibitor, bortezomib and the NF-κB inhibitor, BAY11-7082 , 30. In In a broad range of morphologic and cytogenetic subtypes of primary human AML cells and human AML cell lines, both cytarabine and daunorubicin consistently induce HO-1 expression by 24 h. We also examined the effects of daunorubicin and cytarabine on HO-1 expression at earlier time points finding no increase in HO-1 mRNA levels (not shown). This time lag between addition of drug and HO-1 induction in AML cells is interesting. We hypothesise that this is likely to be due to the complex nature of the HO-1 promoter and its regulation. Nuhn et al have reported that HO-1 expression is usually increased in human cancer cells and is also increased further by the addition of cytotoxic agents . SubsequProtection from apoptosis conferred to AML cells by HO-1 in the current study appears to be medicated through regulation of ROS produced in response to cytarabine and daunorubicin exposure. With regards to HO-1 and its ability to protect cells from undergoing apoptosis, there are a number of different proposed mechanisms. The first is by decreasing the levels of the pro-oxidant, haem , 33; theIn our previous study we showed that basal levels of HO-1 are low compared to normal cells and that NF-κB activation reduced basal HO-1 expression. Furthermore, Nrf-2 activation could override this regulation . In factIn summary we report that silencing HO-1 significantly increases in vitro sensitivity of AML cells to cytarabine and daunorubicin, two front-line chemotherapy agents currently widely used to treat this type of leukaemia. Previously we have shown that HO-1 plays an important role in protecting AML cells from apoptotic stimuli and here we propose that this is likely to be clinically relevant and thus HO-1 warrants further investigation as a target in future therapeutic strategies.The AML-derived cell lines THP-1 and U937, were obtained from the European Collection of Cell Cultures, where cell identity is confirmed by cytogenetic analuysis. Cells were used in the laboratory for a maximum of 6 months post-revival from liquid nitrogen storage, to ensure integrity and retention of characteristics. Anti-human HO-1 antibody was purchased from R & D Systems . All other antibodies were obtained from Abcam . All primers were purchased from Invitrogen . Micro RNA (miRNA) vector was cloned on site. Annexin-V and propidium iodide (PI) were purchased from Abcam . Cytarabine  and daunorubicin  were both purchased from Sigma-Aldrich . All other reagents were purchased from Sigma- Aldrich unless stated.2 in RPMI-1640 from Invitrogen  supplemented with 10% foetal bovine serum (FBS)  and 1% L-glutamine incubated. 293FT cells were obtained from Invitrogen and were grown in DMEM high glucose (Invitrogen) supplemented with 10% FBS, 6 mM L-glutamine, 0.1 mM non-essential amino acids and 1 mM sodium pyruvate.Primary AML cells were obtained under local ethical approval (LREC ref 07/H0310/146). For primary cell isolation, heparinised blood was collected from volunteers and isolated by Histopaque  density gradient centrifugation. U937 were cultured in a humidified atmosphere at 37°C with 5% CO6 cells using RNA lysis solution and a nucleic acid Prepstation both purchased from Applied Biosystems  following the manufactures instructions. Applied Biosystems RNA PCR core kit was used for reverse transcription. SYBR green technology (Roche) was used on cDNA produced via the reverse transcription of purified RNA. After pre-amplification (95 °C for 2 min) the reactions were amplified for 45 cycles  on a Lightcycler 480 (Roche) as described [Total RNA was extracted from 1 × 10escribed . HO-1 mR6 cells using radioimmunoprecipitation assay (RIPA) buffer as described [Total protein was extracted from 1 × 10escribed . ProteinFlow cytometry for measuring apoptosis was performed on an Accuri C6 flow cytometer (Accurri). Samples were collected, centrifuged at 500 × g, 5 min then resupended and stained with Annexin-V and PI dye, followed by detection. N-acetylcysteine (NAC) antioxidant ROS-scavanger was used to assess the presence of ROS, whereby cells were treated with NAC with relevant chemotherapeutic agent for 24 h, prior to sample flow cytometry analysis.2DCFDA) for 15 min at 37 °C and 5% CO2. Flow cytometry was used to measure the fluorescence intensity of H2DCFDA. The mean channel fluorescence of a treated sample is divided by that of the control and then multiplied by 100, thus obtaining the relative percentage change. The relative fluorescence intensity of H2DCFDA detected represents the steady state of ROS generation [A dichlorofluorescein (DCF) assay was used to determine cellular ROS generation . Cells wneration , 36, 37.www.invitrogen.com/rnai). pcDNA™6.2-GW/EmGFP-miR-neg plasmid from Invitrogen was used as a source for negative control miRNA expression. miRNA-encoding viruses were produced with Block-iT Lentiviral Pol II miRNAi expression system (Invitrogen) in 293FT cells. Lentiviral stocks were concentrated using Lenti-X™ concentrator and titers were determined with Lenti-X™ qRT-PCR titration kit . For infection, cells were plated onto 12 well plates at 2.5 × 105 cells/well and infected with lentiviral stocks at an MOI of 10 in the presence of polybrene, then analysed by flow cytometry, RT-PCR and Western blotting 48 h post infection. Stably transduced cells were selected using Blasticidin selection marker (8 μg/ml) for 2 weeks before use.MicroRNA (miRNA) sequence miRNA-HO-1-166 (5′-TCCTCATGAACTCAGCATTCT-3′) targeting human HO-1 was selected with Invitrogen Block-iT RNAi designer software . Values represent the means ± SEMs from at least three independent experiments, or of representative blots/plots that are typical of at least three independent findings.


Im) were recorded in whole-cell/perforated patch clamp configuration. Perfusion of the cells in an external K+ aspartate- (KAsp) solution containing 500 μM cAMP and no extracellular Ca2+, yielded a five to sevenfold increase in the Im current level. The SpIH current increase was associated with detectable hemichannel current activity. Depolarization of cells in Ca2+-free NaCl perfusate with 500 μM cAMP also induced a SpIH current increase. Elevating extracellular Ca2+ to mM levels inhibited hemichannel activity. Perfusion with a depolarizing KAsp solution containing 500 μM cAMP and 2 mM Ca2+ did not increase SpIH currents. The addition of the gap junction blocker carbenoxolone to the external solution inhibited cAMP uptake. Both cell depolarization and lowered extracellular Ca2+ increase the open probability of non-junctional hemichannels. Accordingly, the SpIH current augmentation was induced by the uptake of extracellular cAMP via open membrane hemichannels in Cx43 and Cx26 expressing cells. The data presented here show that hemichannels of Cx43 and Cx26 are permeable to cAMP, and further the data suggest that hemichannels are, in fact, a potential pathway for cAMP mediated cell-to-cell signaling.Cyclic adenosine monophosphate (cAMP) is a well-known intracellular and intercellular second messenger. The membrane permeability of such molecules has potential importance for autocrine-like or paracrine-like delivery. Here experiments have been designed to demonstrate whether gap junction hemichannels, composed of connexins, are a possible entrance pathway for cyclic nucleotides into the interior of cells. HeLa cells stably expressing connexin43 (Cx43) and connexin26 (Cx26) were used to study the cyclic nucleotide permeability of gap junction hemichannels. For the detection of cAMP uptake, the cells were transfected using the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH) as the cAMP sensor. SpIH derived currents ( The extracellular release of cAMP is known to exert effects such as receptor expression in renal cells  and inhiAs a first step in assessing hemichannels as a potential delivery pathway, HeLa cells were transfected with cyclic nucleotide sensor SpIH in order to investigate the permeability of cAMP of unopposed connexin43 (Cx43) and connexin26 (Cx26) hemichannels.2 incubator (5% CO2/95% ambient air). Culture conditions for these cells have been previously published in complete detail , supplemented with 10% fetal calf serum , 100 mg/mL streptomycin (Gibco BRL), and 100 U/mL penicillin (Gibco BRL). The medium also contained 100 mg/mL hygromycin (Sigma) or 1 mg/mL puromycin (Sigma). The cells were passaged weekly, diluted 1:10, and kept at 37°C in a COe detail , 2001. EHeLa cells expressing Cx26 and/or Cx43 were grown on coverslips and stained as described earlier . Commerc+ aspartate- 120; NaCl 10; CaCl2 2; HEPES 5 (pH 7.4); glucose 5; 2 mM CsCl, BaCl2 and TEA+ Cl- were also added. For the Ca2+-free (0 Ca2+) bath solution CaCl2 was omitted. For the regular modified Tyrode external solution (NaCl), K+ aspartate- in the superfusate was replaced with an equal molar concentration of NaCl. The patch pipettes were filled with solution containing (mM): K+ aspartate-, 120; NaCl, 10; MgATP, 3; HEPES, 5 (pH 7.2); EGTA, 10 (pCa ~8); filtered through 0.22 μm pores. In perforated patch experiments, the pipette solution contained 30–50 μM β-escin . During experiments, the cells were superfused with depolarizing bath solution (KAsp) at room temperature (~22°C) containing (mM): K β-escin . The serCyclic AMP transfer through gap junction hemichannels was investigated using single HeLaCx43 and/or HeLaCx26 cells. For the detection of cAMP uptake, the cells were transfected with the cAMP sensor, a cyclic nucleotide-modulated channel from sea urchin sperm SpIH;. Product+ aspartate- (KAsp) bath solution (with 2 mM Ca2+ or Ca2+-free). For cAMP uptake experiments cAMP (Sigma-Aldrich) was added to the external bath solution to reach a concentration of 500 μM. The SpIH derived currents (Im) were recorded from the single cell expressing SpIH and Cx43 and/or Cx26. In some experiments 50 μM of cAMP was introduced via the patch pipette directly in to the cell. In another series of experiments 500 μM cAMP was also locally introduced to the cell membrane via the external pipette. To prevent cAMP degradation a membrane-permeable phosphodiesterase inhibitor IBMX  was added to the bath solution. An adenylate cyclase inhibitor, 2′,5′-dideoxyadenosine  was added to the pipette and bath solutions to inhibit intracellular cAMP production.Wild-type, Cx43 and Cx26 transfected cells were incubated in either in NaCl or KVoltage and current signals were recorded using patch clamp amplifiers (Axopatch 200). The current signals were digitized with a 16 bit A/D-converter  and stored within a personal computer. Data acquisition and analysis were performed with pClamp9 software (Molecular Devices). Statistical analysis was performed using SigmaStat (Jandel Scientific). The Mann–Whitney Rank Sum test was used for all cases unless otherwise noted. The results are presented as means ± SEM.Figure 1, typical punctate staining (in green) at the cell to cell contact regions and the cell membranes of single cells indicates Cx43 (A) and Cx26 (B) expression in HeLa cells stably transfected with Cx43 and Cx26, respectively.HeLa cells stably transfected with mCx43 and hCx26 were immunostained with anti-Cx43 and anti-Cx26 antibodies, respectively. Immunofluorescent staining verified protein expression and localization of Cx43 and Cx26 within the cells  (A) along with the two resultant membrane currents (Im) recorded from the same HeLaCx43 cell transfected with SpIH (B and C). The cell was perfused with the external NaCl (modified Tyrode) solution containing 2 mM Ca2+ and 500 μM cAMP. Figure 2B (left panel) shows whole-cell (perforated patch) currents recorded from a SpIH transfected cell, while the right panel demonstrates experiment conditions. Figure 2C (left panel) shows the current recording from the same cell after the second pipette with 50 μM cAMP in whole-cell mode was attached to it (shown in the right panel). Voltage pulses delivered from a holding potential of 0 mV to test potentials between -20 and -120 mV produced time- and voltage-dependent inward and tail currents (Vm = +50 mV) in SpIH transfected cells. When cAMP was present in the pipette solution, SpIH transfected cells exhibited larger currents. The tail current densities were measured after voltage step to Vm = -100 mV. On average, intracellular application of cAMP increased the peak current level ~fivefold, in comparison to the SpIH transfected cells not treated with cAMP . These results are consistent with those found in a previous report (s report .2+) containing 500 μM cAMP. The left hand panels in Figure 3B show the recorded response of SpIH whole-cell (perforated patch) currents to hyperpolarizing voltage . The recordings were obtained from HeLaCx43 cell perfused with KAsp bath solution with 2 mM Ca2+. On average, Im measured from 10 preparations yielded 6.4 ± 1.1 pA/pF. Figure 3C (left panel) illustrates that the SpIH current increased significantly (~sixfold) in the absence of external Ca2+ . The current amplitudes recorded in the presence and absence of external Ca2+ are summarized in Figure 3D. The SpIH current increase is consistent with the opening of Cx43 hemichannels in Ca2+-free solution and cAMP flux into the cell following the activation of SpIH channels . When the NaCl external bath solution was replaced with KAsp bath solution with no Ca2+, SpIH current increased more than sevenfold . An example of the activation of the SpIH current switching from NaCl bath solution with Ca2+ to the KAsp solution lacking Ca2+ is shown in Figure 4D. Currents recorded from HeLaCx43 SpIH-expressing cells were derived in response to voltage pulses of -100 mV and returning to a tail potential of +50 mV from a holding potential of 0 mV. At approximately the 30-s time mark, perfusion with Ca2+-free KAsp solution was initiated. SpIH currents started to increase over time to a new steady-state value due to cAMP diffusion from the external bath into the cytoplasm via membrane hemichannels. On average, derived from seven preparations, switching NaCl bath solution with Ca2+ to the KAsp solution with no Ca2+, SpIH currents increased from 5.8 ± 0.9 to 37.1 ± 3.6 pA/pF .2+-free KAsp solution with 500 μM of cAMP . Figure 5B shows no current increase when the external NaCl bath solution with 2 mM Ca2+ was exchanged with Ca2+-free KAsp solution, which is in contrast to the considerable current increase when such a procedure was performed on cells expressing SpIH .Experiments performed with HeLaCx43 cells lacking SpIH expression revealed negligible membrane currents in Ca2+ in the external solution, because of opening non-junctional hemichannels and following cAMP diffusion into the cell. These observations are consistent with concept that hemichannels activity is regulated by extracellular Ca2+ . Cell depolarization from the resting potential (~-40 mV) to 0 and +50 mV similarly enhanced the SpIH currents in three other preparations. Due to the different holding voltages, the SpIH currents in each cell were analyzed individually. However, in each of the four cells, regardless of the holding potential, depolarization yielded a significant SpIH current increase over the time from 2.2 to 5.1-fold (~fourfold on average in four preparations). In contrast, there was no significant SpIH current increase detected when the cells were held at a hyperpolarizing voltage of -40 mV. Not only did lowered extracellular Ca2+ increase channel activity, but also depolarization caused activation of hemichannels, i.e., more cAMP flux to the cell and subsequent SpIH current increase. An example of a SpIH current increase and associated detectable Cx43 hemichannel activity is demonstrated in Figure 6B, where the area circled in red denotes the time-dependent current increase, which implies activation of hemichannels during depolarization.The membrane potential controls hemichannel activity and cell depolarization subsequently triggers the opening of unopposed membrane hemichannels .An exampFigures 6, HeLa cells expressing hCx26 also demonstrated cAMP uptake. Figure 7 shows a schematic of the experimental conditions (panel A) and SpIH current recordings from a HeLaCx26 cell transfected with SpIH (panel B). In five preparations SpIH currents increased from 6.4 ± 2.2 to 32.3 ± 3.3 pA/pF when the NaCl external solution with 2 mM Ca2+ was replaced with Ca2+-free KAsp, due to cAMP flux from the external solution through opened membrane hemichannels . In four preparations, some HeLaCx26 cells did not exhibit such SpIH current increases when perfused with Ca2+-free KAsp solution containing 500 μM cAMP . Such an absence of a current increase could be explained by the lack of the sufficient numbers of operational non-junctional hemichannels in the cell membrane. It has been reported that homotypic Cx26 gap junction channels are~7 times less permeable to cAMP than Cx43 channels  there are one or two operational hemichannels with ~280 pS unitary conductance detectable in the cell. This suggests that cAMP flux through one or two operational hemichannels is not enough to induce a SpIH current, in contrast to the example in Figure 7B where presumably there is a much higher number of open hemichannels in Ca2+-free solution. Furthermore, phosphodiesterase activity, with such a small number of operational channels, cannot be ruled out completely. Unitary conductance of hemichannels resolved from the record in Figure 8A, i.e., ~280 pS, corresponds to unitary conductance of Cx26 hemichannels shown in Figure 8B, where the hemichannel current recording was obtained from a HeLaCx26 cell in response to biphasic 50 mV voltage pulses. Discrete steps indicative of the opening (depolarization pulse) and closing (hyperpolarization pulse) of hemichannels are present. The current histograms yielded unitary conductances of 290–300 pS for Cx26 hemichannels, which is in good agreement with Cx26 unitary hemichannel conductance data obtained from Xenopus oocytes  solution with 2 mM Ca2+. The currents were recorded with the patch pipette in whole-cell mode. The second perfusion pipette was brought in close proximity to the cell and some positive pressure was applied allowing the contents of the pipette to reach the cell membrane. Figure 9B shows the current recording for when the perfusion pipette was filled with NaCl solution containing 2 mM Ca2+ and 500 μM cAMP. The current amplitude did not change significantly during local perfusion. However, when the perfusion pipette was filled with depolarizing Ca2+-free KAsp solution and 500 μM cAMP, SpIH currents increased significantly, indicating a cAMP influx through open hemichannels . Figure 9D shows averaged current amplitudes recorded before and during local cAMP application.Perfusion of the cells with CaFigure 10A) with depolarizing KAsp solution  did not induce a SpIH current rise in HeLaCx43/SpIH cells . Connexin deficient HeLa parental cells transfected with SpIH likewise did not exhibit a SpIH current increase during local perfusion with cAMP .To prove that cAMP flux is via hemichannels, similar experiments were performed with the presence of the gap junction channel blocker carbenoxolone (200 μM) in the external bath solution. Local perfusion (These experiments show that membrane hemichannels can be activated over the small area of the cell membrane and allow passage of cAMP into the cell.The data presented here show that hemichannels or connexons of Cx43 and Cx26 are permeable to cAMP as demonstrated by the cAMP sensor SpIH. The data address one essential question: are hemichannels to be considered an alternate pathway for autocrine and paracrine delivery? That is, hemichannels are permeable to one signaling molecule known to be involved in paracrine functions utilizing the extracellular pathway .2+ to allow easy demonstration of hemichannel currents. When extracellular Ca2+ is in the μM range, macroscopic or multichannel data has revealed that hemichannels have a very high open probability with depolarization (One issue that has yet to be clearly addressed is the true operational limits of hemichannels. Previous works have often used lowered extracellular Carization .2+ levels and/or membrane depolarization have the potential to allow both the influx and efflux of solutes. Consistent with this notion is activation of hCx26 hemichannels at voltages above -40 mV (Ischemia is known to result in membrane depolarization of cardiac myocytes  and celle -40 mV . In the 2+ is between 1 and 2 mM, the expected range for the interstitial space. What remains to be determined is the open probability of hemichannels versus extracellular Ca2+ while utilizing conditions to silence other channel types, i.e., K+ channels. For a cell with 10000 hemichannels on its surface and with an imaged open probability of 1% in normal extracellular Ca2+ the number of functioning channels would be approximately 100 open channels at any instant in time. Assuming the cAMP/K+ permeability ratio for Cx43 and Cx26 hemichannels is similar to their respective gap junction channels then the flux per channel is as previously reported (The large conductance and permissive selectivity of Cx43 and Cx26 would also allow significant delivery of solutes like cAMP, possibly with even extremely low open probabilities on the order of 0.1–1%. In fact, little is known about the activity of hemichannels when extracellular Careported , approxiUnderstanding the potential role of hemichannels, unrelated to their precursor role in the formation of gap junction channels, as a potential autocrine/paracrine signaling pathway is a real challenge. It is challenging both from a biophysical perspective and the assessment of autocrine and paracrine effects within tissues. Open probability versus calcium is an important biophysical parameter to clearly define, but it is also necessary to test autocrine/paracrine delivery mediated by hemichannels without an endosomal/vesicular background. The latter can be accomplished using drugs that inhibit or block endosomal/vesicular traffic. This study focused on hemichannels composed of connexins but an equally plausible hemichannel construct is a hemichannel composed of pannexins . Thus, iIn this study, the SpIH gene, which is a cyclic nucleotide gated channel, was used to assess the permeability of hemichannels to cAMP. This is a useful method, which allows accurate estimates of cyclic nucleotide flux via different membrane hemichannels. In such cases, it is the most suitable approach for quick screening of connexin mutants. Defining the permeability and selectivity properties of hemichannels are important factors in understanding their potential role in normal cell physiology and disease states with connexin mutations.A final question posed is if there is a role for autocrine and paracrine delivery mediated by hemichannels within the myocardium? Presently autocrine and paracrine functions for hemichannels are open to debate, but there is strong evidence that cAMP influx can act to reduce the sodium current in cardiac myocytes . AutocriA number of reports reviewed inThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The latter results in oscillatory Ca2+ influx through voltage-gated Ca2+ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration  during these cycles is poorly understood. Importantly, the activation of Ca2+-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca2+ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic  or green fluorescent protein-based recombinant-targeted  probes for Ca2+ and cytosolic ATP/ADP  alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca2+ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca2+ oscillation frequency as stable increases in [Ca2+]mit and cytosolic ATP/ADP; (3) mitochondrial Ca2+ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca2+]cyt and [Ca2+]mit is essentially unaffected by changes in endoplasmic reticulum Ca2+ ([Ca2+]ER). Our findings thus highlight new aspects of Ca2+ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive KThe online version of this article (doi:10.1007/s00424-012-1177-9) contains supplementary material, which is available to authorized users. Increas glucose , 25 or o glucose  have als glucose , 42. The glucose .2+ in multiple compartments simultaneously in the same living cell, whilst measuring (or manipulating) membrane potential through the patch pipette cyt and [Ca2+]mit during temporally complex changes in the former nor the relationship between cytosolic Ca2+ and Ca2+ in other organelles, e.g. the endoplasmic reticulum, was explored.We have recently developed an approach to monitoring intracellular free Ca pipette , 53. ThuFura red , to moniectively . LikewisPerceval  can be ueval  oscillations on the concentration of this ion within the endoplasmic reticulum and assessed whether fluxes between the Ca2+ and the ER may influence [Ca2+]mit dynamics.We therefore sought here to determine how oscillations in electrical activity, and hence cytosolic Ca2+]mit increases, achieved by silencing MCU, inhibits insulin secretion, demonstrating the importance of mitochondrial calcium accumulation for the normal regulation of hormone release from primary β cells. We then demonstrate that the amplitude of the [Ca2+]mit increases displays a remarkable dependence upon the frequency of [Ca2+]cyt oscillations. Thus, “frequency-amplitude decoding” of the oscillations by mitochondria allows the modulation of ATP/ADP production whilst bypassing the need for stable, and potentially damaging, increases in [Ca2+]cyt.We show that attenuation of ER, an adenovirus encoding D4ER was used  in mitochondria and in the cytosol was performed essentially as described [2+] was measured simultaneously in the ER and cytosol using D4ER cameleon and Indo-1 (Invitrogen). 2mt8RP, Fura-Red and Indo-1 were examined at single excitation and emission wavelengths. Either dye was dissolved in DMSO (4 mM) containing 4 % F127-Pluronic. Cells were loaded by incubation with either dye (4 μM) in the extracellular solution for 30 min. Imaging experiments were performed on an Olympus IX-71 microscope with a UPlanFL N × 40, NA 1.2 objective. For acquisition, an F-View-II camera and MT-20 excitation system equipped with a Hg/Xe arc lamp were used, under control of Cell^R software (Olympus). The excitation/emission wavelengths were (nanometres): 410 of 535 (2mt8RP), 490 of 630 (Fura-Red), 490 of 535 , 440 of (465 and 530) (D4ER cameleon) and 350 of 465 (Indo-1). In all cases, cells were maintained at 3 mM glucose for 15–30 min prior to stimulation at the indicated glucose concentrations. Images were acquired at a frequency of 0.2 Hz  and “Ca2+-max” (5 mM Ca2+). The acquisition of the fluorescence and electrophysiological data was synchronized using TTL pulse.Simultaneous imaging of free  and [ATP/ADP]cyt, in response to a glucose step from 3 to 10 or 16.7 mM. After the onset of electrical activity, the mode was switched to voltage clamp to control Vm and impose electrical stimulations. Vm was held at the value of −70 mV. Electrical stimulation was imposed to mimic the naturally occurring bursts of action potentials and comprised 5-s depolarization trains to −30 mV containing 25 ramps of 100 + 100 ms to 0 mV and back . The pipette tip was dipped into pipette solution and then back-filled with the same solution containing 0.17 μg/ml amphotericin B. Series resistance and cell capacitance were compensated automatically by the acquisition software. Recordings, triggered by the TTL pulse, were started in current-clamp mode, and the depolarization of the plasma membrane was monitored simultaneously with cyt whilst [Ca2+]mit was increased only after a much longer lag .The above observations suggested that the degree of Ca2+]cyt changes of a specific frequency cannot readily (i.e. reproducibly between different single β cells) be imposed through the modulation of glucose concentrations alone. Moreover, step increases in glucose concentration would in any case be expected to enhance glycolytic and oxidative metabolism though a substrate supply effect. This, in turn, should increase respiratory chain activity and mitochondrial ATP synthesis independently of any action of Ca2+ on intramitochondrial metabolism. To avoid these confounding effects, we therefore imposed [Ca2+]cyt oscillations of varying frequency by manipulating the plasma membrane potential, and hence the activity of voltage-gated Ca2+ channels, using voltage clamp.In practice, cyt, reflecting influx across the plasma membrane , alone , consistent with a requirement for sustained elevation in glucose metabolism and ATP synthesis for intracellular Ca2+ homeostasis during the above protocols.Similar data to those above were obtained at 10 mM glucose Fig. , whereas2+]mit to stimulation by depolarising trains of different frequencies. The contribution of the each [Ca2+]mit peak to the maximum [Ca2+]mit amplitude achieved after the firing of five bursts , the increase in [Ca2+]mit provoked by the first burst represented ∼70 % of maximum. By contrast, at the highest frequency tested (10 min−1), the first burst achieved ∼30 % of the maximum [Ca2+]mit increase. The values at intermediate burst frequencies lay between these extremes. Thus, the magnitude of the final [Ca2+]mit increase was dependent on the summation of individual [Ca2+] increases that failed to relax to the baseline between depolarisation bursts.To probe further the properties of the frequency-sensing mechanisms of mitochondria, we analysed in detail the response of cyt oscillation frequency to those for the mitochondrial changes measured above. ER Ca2+ ([Ca2+]ER) was therefore measured using the probe D4ER cyt oscillations at low frequency (1.0 min−1) had no discernible effect on the basal [ATP/ADP]cyt  changes in cytosolic Ca2+. To this end, we deployed our recently developed system mit in β cells are governed by the frequency of electrical bursting. Together with our earlier findings  signals could not routinely be achieved. Nonetheless, post-experiment treatment with ionomycin, in high Ca2+-containing solutions, was used to determine whether the probes were likely to have reached saturation. From these trials, and previous calibration of the mitochondrially targeted probe mit levels extended into the low micromolar range at during peaks [2+]cyt (<1 μM).We would stress that here, and in our earlier studies , 53, preed probe , which hng peaks , exceedi2+ with the membrane-bound probe ZIMIR  and consequently cytosolic ATP/ADP. Consistent with our recent findings [2+]mit occurs in the dissociated mouse β cells  action potentials barely affect [Ca2+]mit . Secondly, it may mitigate the longer-term effects of supraphysiological stimulation, e.g. with sulphonylureas, a process akin to the excitotoxicity which leads to excessive Ca2+ influx and eventual cell death in neurons cyt were closely tracked by those in NAD(P)H fluorescence when the frequency of [Ca2±]cyt oscillations remained low . By contrast, high-frequency cytosolic Ca2± oscillations (e.g. at 11.2 mM glucose for β cells) induced the confluence of the individual NAD(P)H spikes and a stable elevation of this parameter. These earlier, and the present study, thus support the view that frequency of oscillations in cytosolic Ca2+ regulates the amplitude of the changes in fuel metabolism by mitochondria, adding to the list of cellular processes, such as gene expression in immune cells cyt.Earlier evidence for the regulation of mitochondrial oxidative metabolism by cytosolic Caatocytes , where mlleagues  were alsne cells , which a2+ increases during repeated cytosolic Ca2+ pulses  cyt increases than those in the mitochondria  that mitochondria in these cells integrate cytosolic pulses to modulate [Ca2+]mit changes and ATP synthesis. Given the impairment in the normal pulsatility in insulin secretion observed in type 2 diabetes cyt and [Ca2+]mit were measured with Fura-Red and 2mt8RP, respectively. The trace is a representative of n = 10 cells. (JPEG 66 kb) (JPEG 58 kb)High resolution image (TIFF 1728 kb)Figure S22+ changes in the cytosol and mitochondria. A A single β cell was bathed in EC containing 10 mM glucose, with Vm being held at −70 mV. The depolarisations were then imposed using the patch pipette, with different frequencies being applied in random order. Glucose in the bath was then changed to 16.7 mM, as indicated, and the depolarisations were applied again. B Effect of the burst frequency on the [Ca2+]cyt (solid red) and [Ca2+]mit (solid blue), measured in 10 mM  and 16.7 mM glucose , measured in the same cell at the same glucose concentration. (JPEG 59 kb) (JPEG 78 kb)Effect of extracellular glucose on free CaHigh resolution image (TIFF 1830 kb)Figure S3Subcellular localisation of D4ER. Epifluorescence images of a cluster of three cells expressing D4-ER (2 days post infection), pre-incubated in Indo-1 (30 min). (JPEG 58 kb) (JPEG 19 kb)High resolution image (TIFF 3071 kb)Figure S42+-ATPase (SERCA) does not cancel the “summation” of cytosolic Ca2+ increases by mitochondria. A Cyclopiazonic acid (CPA) inhibits the sequestration of Ca2+ into ER induced by depolarization. Data are representative of n = 5 traces. Cells were bathed in 16.7 mM glucose, and depolarisations were imposed using the patch pipette. [Ca2+]ER (green trace) and [Ca2+]cyt (red trace) were reported by D4-ER and Indo-1, respectively. B Effect of CPA on the depolarisation-induced dynamics of [Ca2+]cyt and [Ca2+]mit. Data are representative of n = 5 traces. The cell was bathed in the EC solution containing 16.7 mM glucose, and [Ca2+]cyt (red trace), [Ca2+]mit (blue trace), and Vm (upper trace) were monitored/manipulated simultaneously. The cell was voltage-clamped at Vm = −70 mV, and the depolarisation protocol  (JPEG 66 kb)Inhibition of sarco(endo)plasmidic reticulum CaHigh resolution image (TIFF 1776 kb)Figure S52+ entry into β cell mitochondria is favoured during influx from the extracellular space compared to mobilisation from intracellular stores. A Increases in [Ca2+]cyt and [Ca2+]mit induced by depolarization with KCl (upper) and application of acetylcholine . B The inter-dependence between [Ca2+]cyt and [Ca2+]mit increases induced from extracellular solution, by depolarisation with 7.5, 10, 12, 15, 20 and 30 mM KCl (red circles), and from the intracellular stores, mobilised by 10−3, 10−2, 0.05, 0.08, 0.1, 1 and 100 μM acetylcholine (green circles). (JPEG 19 kb) (JPEG 59 kb)Apparent CaHigh resolution image (TIFF 1695 kb)Below is the link to the electronic supplementary material.


However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation.A uniform extracellular stimulus triggers cell-specific patterns of Ca The forms of the Ca2+ signals vary from cell to cell, even in clonal populations of genetically identical cells, while individual cells show characteristic and reproducible Ca2+ responses in terms of amplitude, frequency, and shape of the transient as well as the latency of onset of the initial Ca2+ rise 2+ signals have been referred to as cell “fingerprints”.Extracellular stimuli, such as growth factors, hormones, and neurotransmitters, activate an intracellular effector molecule, phosphoinositide-specific PLC, that catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate  changes during agonist-evoked Ca2+ oscillations in HeLa cells using a genetically-encoded fluorescent IP3 sensor IP3R-based IP3 sensor 1 (IRIS-1) 3 dynamics were unexpectedly complex because [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with little or no fluctuation during cytosolic Ca2+ oscillations 2+ increases during agonist application indicated the presence of both Ca2+-dependent and Ca2+-independent components for IP3 generation in this cell type 3 dynamics in the generation of the cell-specific patterns of Ca2+ signals remain elusive.Real-time monitoring of cytosolic IP2+ and the high-molecular-weight G protein Gh. PLC-ε is regulated directly by small GTPases from the Ras family, and its enzymatic activity can be stimulated by subunits of heterotrimeric G proteins and small GTPases from the Rho family. PLC-ζ was identified as a sperm-specific PLC, and shows extremely high Ca2+ sensitivity for its enzymatic activity compared with the other PLC isoforms. PLC-η is suggested to be activated through GPCR stimulation. Although various tissues express multiple PLC isozymes, the effects of the individual PLC isozymes on the generation of Ca2+ signals are poorly understood.There are six families of mammalian PLC enzymes  consisting of 13 isoforms in humans 3 dynamics using IRIS-1 in cells showing different patterns of Ca2+ signals within a clonal population of genetically identical HeLa cells stimulated with histamine. The IP3 dynamics were characteristically different depending on the patterns of cellular Ca2+ signals, suggesting that the heterogeneity in IP3 production contributes to the cell-to-cell variability observed in Ca2+ signals. We combined an RNAi technique with the real-time IP3 imaging technique to identify the PLC isozymes involved in the generation of IP3 production in this cell type, and found that at least two isozymes in the PLC-β family, PLC-β1 and PLC-β4, are involved in histamine-induced IP3 increases in HeLa cells. RNAi-mediated silencing and overexpression experiments for PLC-β1 and PLC-β4 revealed that these isozymes differentially contribute to the determination of the characteristic parameters of Ca2+ signals, i.e. the Ca2+ oscillation frequency and the time constant of Ca2+ spike amplitude decay. These findings provide important clues toward understanding the molecular mechanism that underlies the generation of cell-specific patterns of Ca2+ signals in genetically identical cell populations.In this study, we measured the IP2+ oscillations were accompanied by characteristic IP3 dynamics in individual cells, we performed simultaneous monitoring of [Ca2+] and [IP3] at the single-cell level. Indo-5F was loaded into IRIS-1-expressing HeLa cells and the cells were stimulated twice with 3 µM histamine with an interval of approximately 20 min. Consistent with previous studies 2+ dynamics, which varied in the pattern (sustained oscillations or damped oscillations) and the frequency of Ca2+ oscillations , even within the same microscopic field of view  and 2+ oscillations at 3 µM histamine). To analyze the results quantitatively, we measured the time constants for the progressive decrease in the amplitude of Ca2+ spikes  to 0.3 s−1 (rapidly damped oscillations). There was no obvious dependence of the time constant on the histamine concentration, and individual cells showed similar time constants irrespective of the histamine concentration  and D-cells (blue). The frequency increased depending on the concentration of histamine, with slopes of 2.2±1.1 mHz/µM histamine  for S-cells and 1.2±1.0 mHz/µM histamine  for D-cells. There were large cell-to-cell variabilities in the Ca2+ oscillation frequencies, but the frequencies in S-cells tended to be smaller than those in D-cells (t-test) in the Ca2+ oscillation frequencies between S-cells and D-cells following stimulation with 1 µM histamine  and 3 µM histamine  (2+ spikes and the time dependence of the change in the amplitude of Ca2+ spikes. D-cells . We deteD-cells) . These f2+ spike amplitude on the histamine concentration differed between S-cells and D-cells . To identify the PLC isozymes involved in the generation of IP3 dynamics in histamine-stimulated HeLa cells, total RNA was isolated from HeLa cells and subjected to RT-PCR analyses with specific primer pairs  and a slow gradual increase (2nd component) in response to the combination of histamine receptor stimulation and cytosolic Ca2+ increase  and 71% (siRNA#2) compared with the mean change with the control siRNAs mGC and hGC , 38% (PLCβ1KD siRNA#2), 47% (PLCβ4KD siRNA#1), and 51% (PLCβ4KD siRNA#2) compared with the mean value for the control siRNAs mGC and hGC  were larger than that in control cells  and the fraction of cells showing damped oscillations was increased (2+ oscillation frequencies in PLC-β1 knockdown cells were similar to that in control cells (37.1±8.6 mHz)  . The intwn cells .2+ oscillations with small inverse time constants (less than 10−3 s−1) were detected in some cells, but the overall mean inverse time constants did not differ significantly from that in control cells (2+ oscillations in PLC-β4 knockdown cells (21.1±7.2 mHz for siRNA#1 and 17.0±10.5 mHz for siRNA#2) were significantly smaller than that in control cells (37.1±8.6 mHz)  . The int reduced , similarwn cells , while tse cells . In PLC-wn cells . These f2+ dynamics, we examined cells transfected with PLCβ1- or PLCβ4-IRES-mRFP constructs  was significantly smaller than that in control cells   than that in control cells   . In cont4.5 mHz) . These foduction . Insteadng cells . Taken t2+ changes in response to bath applications of histamine, even in the same microscopic field of view, as reported in other cell lines 2+ signals by the Ca2+ oscillation frequencies and the time constants of Ca2+ spike amplitude decay, and found that a difference in histamine sensitivity could not account for the cell-to-cell variability in Ca2+ signals. A remarkable finding in this study is that the IP3 dynamics are characteristically different depending on the patterns of Ca2+ signals  by replacing the AcGFP1 cDNA with an mRFP cDNA. A full-length mouse PLC-β1 cDNA was obtained by PCR from mouse PLCβ1-pBluescriptII KS(+), and cloned into the EcoRI and BamHI sites of IRES-mRFP. A full-length mouse PLC-β4 cDNA was obtained from mouse PLCβ4-pBluescriptII KS(+) by XhoI digestion, and inserted into IRES-mRFP.2.HeLa cells were maintained in DMEM  supplemented with 10% FBS at 37°C under 5% COm values to maintain uniform amplification efficiency. Primers for β-actin were designed under similar criteria and used as an internal control. The PCR fragments were analyzed by 2% agarose gel electrophoresis.Total RNA was extracted from HeLa cells using a TRIzol Reagent Kit (Invitrogen), and reverse-transcribed using random primers and SuperScriptII reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. cDNA fragments for the 13 PLC genes were amplified by PCR using specific primer sets . The priTotal HeLa cell lysates were prepared by direct lysis with SDS sample buffer  and subjected to electrophoresis. The separated proteins were transferred onto PVDF membranes (Millipore), and the PLC isozymes and β-actin were probed with specific primary antibodies. After treatment with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies , the antibody-bound proteins were detected with chemiluminescence reagents  and a LAS4000mini system (Fujifilm).2 chamber, the siRNA solution was removed by replacement with 1.5 ml of fresh culture medium.Fresh culture medium (0.8 ml) was supplied to HeLa cells cultured in 35-mm dishes at 30 min prior to transfection. Next, 1 µl of 20 µM siRNA solution and 1 µl of LipofectAMINE 2000 (Invitrogen) were independently diluted in 100 µl of Opti-MEM (Invitrogen), and the two mixtures were combined and incubated for 20 min at room temperature. The mixed solution was added to the cells to give a final concentration of 20 nM siRNA. After incubation for 8–12 h in a CO2, 2 mM CaCl2, 10 mM glucose) as an imaging medium at 37°C. For experiments with artificial control of [Ca2+], cells were pretreated with 1 µM thapsigargin in the absence of extracellular Ca2+ to deplete the cytosolic Ca2+ stores by blocking sarcoplasmic or endoplasmic reticulum Ca2+-ATPase pumps. Imaging was performed at 37°C using an inverted microscope  with a cooled charge-coupled device camera  and a 40× (NA 1.35) objective. A 330–348-nm excitation filter and 460–510-nm emission filter and a 425–445-nm excitation filter and a pair of 460–510-nm (cyan) and 525–565-nm (yellow) emission filters were used for the fluorochromes Indo-5F and IRIS-1, respectively. An emission splitter  was used with a fast light source exchanger . Sequential excitation of IRIS-1 and Indo-5F was performed using a 450-nm dichroic mirror and two excitation filters (425–445-nm filter for IRIS-1 and 330–348-nm filter for Indo-5F). Dual emission at 460–490 nm (for IRIS-1 and Indo-5F) and >520 nm (for IRIS-1) was split with a 460–490-nm filter, a long-path 520-nm barrier filter, and two 505-nm dichroic mirrors equipped in W-view. Images were acquired at 0.5 or 1 Hz for measurements of Ca2+ oscillations in intact cells and at 0.17, 0.25, or 0.5 Hz for measurements in thapsigargin-treated cells. Image acquisition was performed with MetaFluor software (Molecular Devices). Spectral analyses of Ca2+ oscillations were performed as described previously For imaging of siRNA-treated cells, HeLa cells at 36–42 h after siRNA transfection were transfected with plasmids containing the IRIS-1 cDNA using a transfection reagent . The cells were subjected to imaging at 24–30 h after the IRIS-1 transfection. For imaging of cells expressing recombinant PLC-βs, HeLa cells were transfected with PLCβ1-IRES-mRFP, PLCβ4-IRES-mRFP, or IRES-mRFP plasmids using the TransIT transfection reagent at 20–36 h before IRIS-1 transfection. After loading the cells with 5 µM Indo-5F AM (Dojindo), imaging was performed under a constant flow (2 ml/min) of balanced salt solution  Ca2+ spike amplitudes were defined as shown in the left scheme. The time constants were estimated by fitting the amplitudes with a single exponential function, as shown on the right. (B) Histogram of the time constants of Ca2+ spike amplitude decay observed in HeLa cells stimulated with 3 µM histamine. The arrow indicates the threshold for dividing the cells into S-cells and D-cells.(PDF)Click here for additional data file.Figure S22+ spike and the IP3 production between S-cells and D-cells.Difference in histamine concentration-dependence of the amplitude of the first Ca (A) Relationships between the histamine concentrations and the peak amplitudes of the first increase in Indo-5F signals (F/F0) in S-cells (red) and D-cells (blue). (B) Mean values of the Indo-5F signal changes shown in (A). Statistical analyses were performed by one-way ANOVA followed by Scheffe’s multiple comparison test. *P<0.05, **P<0.01. NS: not significant. (C) Relationships between the histamine concentration and the integrated IP3 signals observed in S-cells (red) and D-cells (blue). Data represent means ± SD. Statistical analyses were performed using Student’s t-test. *P<0.05.(PDF)Click here for additional data file.Figure S3PLC isozyme-specific knockdown does not affect the expressions of other PLC isozymes. Western blotting analyses of total lysates prepared from HeLa cells treated with PLC isozyme-specific siRNAs. The isozyme-specific antibodies used for the western blotting analyses are shown on the left. The data are representative of at least two independent experiments.(PDF)Click here for additional data file.Figure S4Validation of the IRIS-1 signal changes observed in thapsigargin-treated HeLa cells. (A and B) The maximal IRIS-1 signal changes after addition of 3 µM histamine (A) and 2 mM Ca2+ (B) were plotted against the basal CFP intensity of IRIS-1 (top), resting IRIS-1 C/Y ratio (middle), and integrated value of Indo-5F change for 10 min (bottom). (C) The initial rate of IRIS-1 signal change (1st component) after addition of 3 µM histamine plus 2 mM Ca2+ was plotted against the basal CFP intensity of IRIS-1 (top), resting IRIS-1 C/Y ratio (middle), and initial rate of Indo-5F change (bottom). (D) The maximal IRIS-1 signal changes after addition of 3 µM histamine plus 2 mM Ca2+ (2nd component) were plotted against the basal CFP intensity of IRIS-1 (top), resting IRIS-1 C/Y ratio (middle), and integrated value of Indo-5F change for 10 min (bottom).(PDF)Click here for additional data file.Figure S53] changes after histamine stimulation plus [Ca2+] elevation in thapsigargin-treated PLC-β1 and PLC-β4 knockdown cells.[IP Representative traces of IRIS-1 signal changes (ΔR/R0) after addition of 3 µM histamine  plus 2 mM Ca2+  observed in thapsigargin-treated HeLa cells transfected with control siRNA mGC (left), PLCβ1KD siRNAs (middle), or PLCβ4KD siRNAs (bottom).(PDF)Click here for additional data file.