Nonetheless, the extensive use of different kinds of dandelion extracts and preparations is based on empirical findings. Some of the tissue-specific effects reported for diverse dandelion extracts may result from their action on intracellular signaling cascades. Therefore, the aim of this study was to evaluate the effects of an ethanolic dandelion root extract (DRE) on Ca2+ signaling in human embryonic kidney (HEK) 293 cells. The cytotoxicity of increasing doses of crude DRE was determined by the Calcein viability assay. Fura-2 and the fluorescence resonance energy transfer (FRET)-based probe ERD1 were used to measure cytoplasmic and intraluminal endoplasmic reticulum (ER) Ca2+ levels, respectively. Furthermore, a green fluorescent protein (GFP)-based probe was used to monitor phospholipase C (PLC) activation (pleckstrin homology [PH]–PLCδ–GFP). DRE (10–400 µg/mL) exposure, in the presence of external Ca2+, dose-dependently increased intracellular Ca2+ levels. The DRE-induced Ca2+ increase was significantly reduced in the absence of extracellular Ca2+. In addition, DRE caused a significant Ca2+ release from the ER of intact cells and a concomitant translocation of PH–PLCδ–GFP. In conclusion, DRE directly activates both the release of Ca2+ from internal stores and a significant Ca2+ influx at the plasma membrane. The resulting high Ca2+ levels within the cell seem to directly stimulate PLC activity.Dandelion  Acmella oleracea [Sclerocarya birrea [Agelas clathrodes [Tedania ignis [Taraxacum officinale Weber, known as dandelion, is a perennial weed that has been used for hundreds of years (starting from the 10th and 11th centuries) as a traditional medical remedy for several diseases [To date, natural products from medicinal plants, microorganisms, marine organisms  are in diseases . As a madiseases ,7. Howevdiseases , therefoA possible explanation of such a wide panel of physiological effects might be found in the chemical composition of the different dandelion preparations. Dandelion’s extract might be composed by the whole plant or by different parts of it , alone or in combination. In addition, its chemical composition strictly depends on both the extraction protocol and the solvents used  that have to be properly designed and identified to efficiently produce extracts containing the desired bioactive compounds. For example, the phytochemical composition of dandelion roots extracts (DRE) reported the presence of sesquiterpenes, various triterpenes, phytosterols, and phenolic compounds . Among tT. officinale suggests the ability of cells to respond to its herbal extracts through a basic and widespread intracellular mechanism.These various chemical components may act individually, additively, or in synergy to regulate different tissue-specific functions. Still, the surprising amount of physiological functions that are somehow related to the medical use of 2+ homeostasis. Among the more general transduction pathways of a cell we decided to analyze specifically Ca2+ signaling for two main reasons. First, even though there is no information available in the literature about the effects of dandelion extracts on intracellular Ca2+ dynamics, numerous reports regarding the effects of single bioactive components, especially hydroxycinnamic acid derivatives, have been published. Caffeic acid (CA) and its derivatives are natural phenolic compounds that affect cellular Ca2+ homeostasis in different experimental models, such as human gastric cancer (SCM1) cells [2+-mediated insults by reducing, through cell-specific mechanisms, Ca2+ entry at the plasma membrane [Therefore, in this study, we evaluated whether the acute exposure of HEK293 cells to an ethanolic dandelion root extract (DRE) might impact intracellular Ca1) cells , T lymph1) cells , and Jur1) cells . Chlorogmembrane ,17,18.2+ events . This implies either that the two players  share common cellular targets or that Ca2+ is a dandelion-induced mediator within the cells.Second, most of the physiological functions that are regulated by dandelion extracts are finely modulated by intracellular Ca2+ levels within the endoplasmic reticulum, respectively, in HEK293 cells in response to acute exposure to DRE. In addition, a green fluorescent protein (GFP)-based probe [2+ levels. Such rise in cytosolic Ca2+ levels was due to both Ca2+ release from the endoplasmic reticulum and Ca2+ entry at the plasma membrane, likely via store-operated channels. DRE exposure also induced the activation of a downstream signaling element such as PLC, which is elicited, even though not exclusively, by G-protein-coupled receptors [Therefore, we used Fura-2 and the fluorescence resonance energy transfer (FRET)-based probe ERD1  to measued probe  was usedeceptors .2+ events at the cellular and molecular level will pave the way for the proper use of this promising extract as a natural pharmaceutical tool.The precise dissection of DRE-induced intracellular CaDandelion dried roots were extracted with ethanol by exhaustive maceration. Ethanol is an appropriate solvent for both phenols and other bioactive compounds because of its intermediate polarity. It is well known that solvents affect the extraction of bioactive compounds and the phytochemical profiles of extracts from various plant parts . ConsequThe chemical composition of our dandelion ethanolic root extract (DRE) was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC). p-coumaric acid (Rt: 14.72 min), and ferulic acid (Rt: 17.71 min). All compounds were identified comparing their retention time and UV spectrum with those of reference standards. The other peaks of the extract could not be identified because of a lack of validated reference standards to compare with. All these compounds had already been identified in dandelion root extracts [We also identified, although present in lower amount, gallic acid (Rt: 4.72 min), vanillic acid (Rt: 10.83 min), syringic acid (Rt: 11.32 min), extracts . In thisOn the basis of this result, in the following functional studies, we used DRE at 400 μg/mL (or lower).2+ levels in HEK293 cells. In the presence of 1.2 mM extracellular Ca2+, 400 μg/mL DRE elicited cytosolic Ca2+ transients that were not significantly larger  than those produced by a saturating dose of the Ca2+-mobilizing agonist ATP (100 µM) in the same cells .The results collected in 2+ response induced by DRE was dose-dependent. The smallest, albeit significant, increase in intracellular Ca2+ was detected at a concentration of DRE as low as 10 μg/mL . Concentrations of 50 and 200 μg/mL DRE induced linear increases in intracellular Ca2+ that were, however, significantly smaller than those induced by ATP . In addition, the higher was the concentration of DRE used, the larger was the number of responsive cells on a single coverslip  on the intracellular Ca2+ levels. Both compounds, enriched in our DRE (see 2+ homeostasis [2+ changes were recorded (data not shown). Also, when we exposed HEK293 cells to the highest investigated concentrations of either caffeic acid (150 μM) or chlorogenic acid (200 μM) we did not record intracellular Ca2+ changes . These results suggest that DRE was able to release Ca2+ from internal stores. Indeed, 2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid (CPA) in free extracellular Ca2+ to deplete intracellular Ca2+ stores, the Ca2+ response induced by DRE in control conditions was completely prevented. Note that this experimental approach also revealed that the acute exposure to 400 μg/mL DRE did not interfere with the mechanisms underlining CPA-induced endoplasmic reticulum (ER) Ca2+ release and with the capacitative Ca2+ entry induced by perfusion with 5 mM Ca2+ after store emptying. T. officinale has been usually associated with diuretic effects, thus renal cells represent putative physiological targets. In separate experiments, we showed that exposure to DRE (400 μg/mL) induced Ca2+ transients similar to those obtained in HEK293 cells either in the presence or absence of extracellular Ca2+ also in a renal model of epithelial cells  (data not shown).The Ca2+ release process in more detail in intact HEK293 cells transfected with the FRET-based probe ERD1. 2+-free solution for 4 min decreased the intraluminal free Ca2+ levels by 46.12 ± 6.6% . DRE-induced intraluminal reduction was normalized with respect to the maximal ER-emptying effect elicited by 5 μM ionomycin in the absence of Ca2+. Exposure of cells to the Ca2+-mediated agonist ATP .We next examined the ER Canist ATP b was abl2+ entry at the plasma membrane [2+ in a Ringer’s solution nominally free of Ca2+, in the continuous presence of DRE, induced an increase in the intracellular Ca2+ levels likely through store-operated channels (SOCs). Comparable results were obtained when we used, in the same experimental protocol, ATP instead of DRE.It is widely recognized that the emptying of the endoplasmic reticulum activates store-operated Camembrane . As show2+ stores  or activate a plasma membrane receptor coupled to a Gq alpha subunit (Gαq). However, when we used ionomycin in a protocol similar to that depicted in The results collected so far suggest that DRE can either cross the plasma membrane and exert direct actions on internal Ca2+, considered in this protocol as the control response. The quantal increase  of the extracellular Ca2+ concentration rapidly raised the cytosolic amount of Ca2+ to about 160% in the presence of 10 mM extracellular Ca2+ . When we repeated the same protocol with DRE instead of ionomycin, we recorded, in the presence of 10 mM extracellular Ca2+, smaller increases in cytosolic Ca2+  that suggested a capacitative Ca2+ entry rather than a ionophore-mediated influx through the plasma membrane.2+ mediated by GPCR.Thus, is there a G-protein coupled receptor (GPCR) involved? It is pharmacologically complicated to specifically block Gαq. This is the reason why we decided to characterize features that are generally related to the increase in cytosolic Ca2+ response induced by DRE was significantly decreased when repeated, at the same concentration, after 10–15 min of DRE washout . When we increased the interval between two DRE stimulations, we found a significant recovery of the second Ca2+ response that resulted non-significantly changed compared to the first one after 45 min . One possible explanation could be that it takes time to refill the ER that has been challenged after the first DRE stimulation. However, when we repeated the same experimental protocol with ATP, we found a complete recovery of the Ca2+ response after 15–20 min (2+ recorded under both agonists (DRE and ATP) stimulation is of similar extent and (ii) the Ca2+ homeostatic mechanisms are the same for the two agonists, we conclude that the hypothesis that DRE is activating a GPCR has to be taken into account.First, the Ca5–20 min b. Indeed2+ release from the ER would have been the result of the activation of the Gαq/PLC/IP3 pathway. Therefore, we used 10 µM U73122 in order to inhibit PLC. 2+ levels both in the presence of U73122 and 10 min after its washout. However, these results are significantly affected by the fact that U73122 likely increased intracellular Ca2+ levels by emptying the endoplasmic reticulum, as already shown for baby hamster kidney (BHK21) cells [2+ increase that we observed in our study. Therefore, we cannot use these experiments to extrapolate information about the putative activation of PLC following exposure to DRE.Under this scenario, the Ca1) cells . This seThus, as an alternative strategy, we used a GFP-based indicator, PH–PLCδ–EGFP ,24 that n = 4, m = 63). In order to have a semiquantitative appreciation of the responses, as for the previous experiments, we analyzed only cells which clearly responded to ATP with either a decrease of the plasma membrane signal or the classical antiparallel behavior of the membrane and cytosolic fluorescence intensity.The two typical patterns of response observed in single cell epifluorescence experiments in PH–PLCδ–EGFP-expressing HEK293 cells upon stimulation with ATP and DRE are shown in 2+ release from the endoplasmic reticulum through the formation of IP3 and the activation of IP3R. Ca2+ released from the ER might, in turn, activate the ryanodine receptors (RyRs) showed to be expressed and functional in HEK293 cells [2+ was significantly reduced  under prolonged (at least 50 min) RyRs blockade with high concentrantions of ryanodine (100 µM), clearly indicating Ca2+-induced Ca2+ release as an additional mechanism in DRE-elicited ER Ca2+ release.Thus, DRE-induced activation of PLC might induce Ca93 cells . Of noteT. officinale (dandelion) is an ethnomedical herb used as anti-inflammatory, antioxidant, diuretic, choleretic, laxative, and to treat arthritis and liver disorders [The use of plant extracts to treat diseases is ancient, and popular observations regarding their use and efficacy prompt the investigation of their therapeutic properties . The ideisorders . Howeverisorders .2+ increase in HEK293 cells  and direct comparison with drugs  that have a well-known action on Ca2+ homeostasis. We found that the rapid Ca2+ transient that follows DRE exposure results from a significant Ca2+ release from the endoplasmic reticulum and Ca2+ entry at the plasma membrane probably through store-operated Ca2+ channels. Of note, DRE stimulation induced a significant activation of PLC. This important observation was obtained considering the subcellular localization of a GFP-tagged PH–PLCδ construct [In this study, we showed, for the first time, that acute exposure  to an ethanolic dandelion root extract (DRE) induced a dose-dependent and reversible Ca93 cells . DRE-medonstruct ,24. In tonstruct ,24, thusonstruct .2+ entry and “in situ” Ca2+ release from the ER and activation of PLC that seconds the cytosolic Ca2+ increase. In the second scenario, DRE (or one of its components) activates a G-protein coupled receptor at the plasma membrane and PLC and IP3 production which, in turn, induces Ca2+ release from the endoplasmic reticulum potentiated by Ca2+-induced Ca2+ release from RyRs , was ineffective when used at a concentration (200 µM) proportional to its presence in our DRE. Therefore, regardless of which theory is correct, it is currently unknown if the Ca2+ effects measured in response to DRE are mediated by the whole extract or by individual components diverse from caffeic and chlorogenic acids.Under the same scenario, Chang et al. showed that 800 µM caffeic acid evoked Caeceptors . In addin HEK293  nor in Mn HEK293 . In our 2+ homeostasis at different subcellular locations is an important piece of the puzzle for understanding the wide array of biological functions (of different organs and systems) reported to be modulated by T. officinale. As a matter of fact, and as suggested for Taraxacum [2+ signaling finely regulates processes like cellular proliferation [Taraxacum–Ca2+ interplay can be considered as a starting point to better project and more precisely target the use of T. officinale extracts as pharmacological tools.Whatever the specific mechanism of action at the plasma or intracellular membrane, the direct observation that DRE impacts Caaraxacum ,29,30,31feration , bile seferation , diuresiferation , inflammferation , and oxiferation , just toT. officinale extracts are increasing, and it has been shown that dandelion root extracts are mainly rich in hydroxycinnamic acids, whereas hydroxybenzoic acids derivatives and flavonoids have been reported in lower amounts. In this study, the chemical composition of an ethanol extract of dandelion roots was investigated by RP-HPLC analysis  induced a significant increase in cytosolic Ca2+ levels in HEK293 and MCD4 cells. We proved that spilanthol, the main component of the Acmella extract, acted like a potent and natural diuretic in mouse by activating a mechanism based on Ca2+-induced inhibition of cytosolic cyclic adenosine monophosphate (cAMP) [Scrophularia orientalis [Echinacea extracts [Echinacea, such as alkamides (via activation of cannabinoid receptors) [2+ peaks.DRE is not the first herbal extract tested for an effect on the mechanisms underlying Cae (cAMP) . More exe (cAMP) , Scrophuientalis , and Echextracts ,44. For ceptors)  and unkn2+ homeostasis in HEK293 cells. The mechanism proposed, although not completely characterized, indicates Ca2+ signaling as the cellular mediator used by the cells to transduce the effects of the exposure to the bioactive components of the T. officinale extract. Of note, this important evidence has a number of perspectives. First, researchers know how the mechanisms underlying Ca2+ homeostasis work, how to modulate them pharmacologically, and how they impact diverse biological activities. Thus, it will be possible to finely tune T. officinale effects on Ca2+-signaling in order to improve the biological outcome. Second, Ca2+ signaling intersects the activity of a huge number of other signaling molecules  in terms of their relative composition of bioactive compounds will help to link the presence of specific components to the activation of precise signaling mechanisms. This latter perspective might have an enormous pharmaceutical impact.In conclusion, this report highlights the effect of an ethanolic dandelion root extract on Cag., cAMP ) and/or g., cAMP ). Thus, T. officinale was collected in Basilicata region (40°34′50.9′′ N 15°49′38.2′′ E) in October 2017; the roots were separated from the aerial parts and then dried at room temperature in the dark.w/v  under continuous stirring for 3 days, changing the solvent every 24 h. The extract was filtered, and the solvent was removed in a rotary evaporator. The dried crude extract was kept in the dark until use.The dried roots (15 g) were extracted by maceration with EtOH 95% at 1:10 p-coumaric acid, and gallic acid, were used . The quantification of phenolic compounds was realized by the comparison between chromatogram recorded absorbances and external calibration standards. All experiments were carried out in triplicate.The re-dissolved crude extract (100 mg/mL) was analyzed on an analytical HPLC–DAD  unit using an EC 250/4.6 Nucleodur 100-5 C18ec column . The mobile phase involved two solvents: water–formic acid (5%) (A) and acetonitrile (B). The gradient elution method has already been described by our group . All pea2/95% air. MCD4 cells, a clone of M-1 cells stably transfected with human-Aquaporin (AQP) 2, were cultured as described elsewhere [2+ levels with Fura-2-AM . Alternatively, 24 h after plating, the cells were transfected with ERD1 [HEK293 cells were grown in Dulbecco’s modified Eagle medium (DMEM) Glutamax supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  and were maintained in a humidified incubator at 37°C in the presence of 5% COlsewhere . Trypsinith ERD1  or the Pith ERD1  and usedµL of 1µM Calcein-AM  in phosphate buffered saline (PBS) was added for 30 min at 37°C. The fluorescence was detected by FLEX STATION 3  plate reader using a blue filter .The cytotoxic effect of the crude DRE was determined by the Calcein-AM viability assay . HEK293 2+ recordings, the cells were seeded on 25 (or 18) mm Ø glass coverslips. HEK293 cells were loaded with 2–4 μM Fura-2-AM  for 30 min at 37°C in DMEM Glutamax, followed by 15 min in an extracellular solution to allow Fura-2 de-esterification. The coverslips with dye-loaded cells were mounted in an open perfusion chamber , and recordings were carried out using an inverted Eclipse TE2000-S microscope  equipped for single cell fluorescence evaluations and imaging analysis. Each Fura-2-AM loaded sample was illuminated every 5 s through a 40× oil immersion objective  at 340 and 380 nm. The emitted fluorescence was passed through a dichroic mirror, filtered at 510 nm , and captured by a cooled CCD CoolSNAP HQ camera . Additional technical information about the setup used in our imaging facility is available in other publications from our group [n indicates the number of experiments performed for each protocol, m the number of cells analyzed in n experiments.For cytosolic Caur group ,50. Fluo2+ levels within the endoplasmic reticulum of HEK293 cells transfected with ERD1. Real-time FRET experiments were carried out using the MetaFluor Fluorescence Ratio Imaging Software . FRET from cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP) was evaluated by excitation of CFP (435 nm) and measurement of the fluorescence emitted by YFP. The results are presented as the emission ratio 485/535 nm collected every 5 s in control conditions and after stimulation with the agonists . The emission ratio was normalized to the maximal store emptying induced by 5 μM ionomycin in an extracellular Ca2+-free solution (R/R0). The data from 5 to 10 transfected cells were summarized in a single run and averaged in a plot ± SE, and at least four independent experiments for each agonist were conducted. n indicates the number of experiments performed for each protocol, m the number of cells analyzed in n experiments.The same imaging setup and perfusion apparatus were used for the measurements of Can indicates the number of experiments performed for each protocol, m the number of cells analyzed in n experiments.The synthesis of intracellular InsP3/PLC activation was evaluated with the GFP-tagged pleckstrin homology domain of PLCδ1 (PH–PLCδ1–EGFP) ,27,51. H2, 1 MgCl2, 5 glucose, 10 HEPES, adjusted to pH 7.40 with NaOH.Most of the chemicals were obtained from Sigma . The experiments were carried out with an extracellular (Ringer’s) solution containing (in mmol/L): 140 NaCl, 5 KCl, 1 CaClIonomycin and Fura-2-AM were from Thermo Fisher Scientific . When dimethyl sulfoxide or ethanol were used as a vehicle, the final solvent concentration was always below 0.01% or 0.1%, respectively.t test. The data were expressed as means ± SE with n equal to the number of experimental runs. For Fura-2 and ERD1 ratio imaging experiments, p < 0.05 was considered statistically significant.Whenever possible, responses to DRE and ATP  were compared in the same cell (paired data), thus eliminating concerns about the variability of the starting Fura-2 ratio. Whenever appropriate, paired data were assessed for statistical significance using the Student’s


It is therefore tightly regulated. It has been recently proposed that LETM1—an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis—also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+/Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity.Ca Monovalent cation homeostasis is dysregulated upon LETM1 depletion+/H+ exchange activity is decreased in LETM1 knockdown cellsK+ accumulation in the mitochondrial matrixLETM1 depletion results in K2+ transport modulatorsLETM1 knockdown does not affect expression of major mitochondrial Ca2+ fluxes are dependent on Na+LETM1-regulated mitochondrial Ca+ , the other more promiscuous . Given the ion composition of the cytoplasm and mitochondrial matrix, this latter non-selective antiporter has been generally considered to function as a K+/H+ exchanger (KHE)    . Mitochondrial Ca2+ release has been shown to be induced by Na+ and mediated by a Na+/Ca2+ antiporter, which exchanges mitochondrial Ca2+ for external Na+  , GAPDH . Secondary HRP conjugated AffiniPure antibodies were from Jackson Immunoresearch : goat anti-mouse (115-035-008) and goat anti-rabbit (111-035-008).2O.All reagents were from Sigma Aldrich  unless otherwise indicated. Thapsigargin was from Tocris  and puromycin from Invivogen . All buffers as formulated were completed with MilliQ  ddHPrimers are listed in the Table HeLa cells were a gift from E. Tomasich . All cells were maintained in DMEM (Gibco) supplemented with 10% (v/v) FBS (Gibco) and 1X penicillin/streptomycin/glutamine (Gibco). Cells were regularly screened for mycoplasma (MycoAlert Lonza kit).Cells were transfected with Turbofect  according to manufacturer's instructions; 48 h post transfection the media was changed to growth media with puromycin (2 μg/mL). After a resistant population of cells was established, cells were maintained in growth medium containing puromycin (2 μg/mL).Cells were washed with PBS (Gibco) then harvested using lysis buffer , 1% IGEPAL C360 and one tablet of protease inhibitors with EDTA (Roche) per 10 mL). Cell lysates were vortexed for 30 min at 4°C and then cleared by centrifugation at 15,000 g for 15 min, 4°C. Proteins were quantified by Bradford assay  using BSA as standard. Protein lysates were separated by SDS-PAGE and blotted onto 0.45 μm PVDF membranes . Membranes were immunoblotted as indicated and fat-free milk was used as blocking medium. Blots were quantified using the BioRad Image Lab (v5.2.1) software.Total RNA was isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was DNase I (Invitrogen) digested before reverse transcription using the High-Capacity cDNA Reverse Transcription Kit . Diluted cDNA was then used for quantitative PCR using the GoTaq qPCR master mix  and the reaction was run on the StepOne Plus (AB Biosystems). Analysis was performed using the StepOne Plus software (v2.3) and Expression Suite (v1.0.3).2+ green 5N) or intracellular like buffer II (ICL buffer II) , 10 μM EGTA-Tris, 1 mM KPi, 5 mM malate, 5 mM glutamic acid, 1 μM Ca2+ green 5N).Importantly, these buffers are identical, with the exception that ICL I contains sodium glutamate and ICL II glutamic acid. The pH of the ICL buffers was verified prior to measurements. Sequential additions of 10 μM Ca2+ (as CaCl2), 0.2 μM ruthenium red (RR) or 1.2 μM ruthenium 360 (R360), 10 mM Na+  and 2 μM FCCP were as indicated in Figure 4. Ca2+ uptake/release measurements were performed at 25°C on a LS55 spectrofluorometer (Perkin Elmer) with the following parameters, λex = 505 nm, λem = 535 nm, slit width: 2.5 nm.Cell counting, harvesting and permeabilization were performed as in Wilfinger et al. , with on2+ immediately prior to measurement by incubation with 1 μM A23187 and 1 mM EDTA to eliminate Mg2+, the blocker of the KHE. To measure KOAc- or NaOAc-induced passive swelling, KOAc  were harvested and permeabilized as described in Wilfinger et al. , HEPES (pH 7 and 7.4) or TRIS (pH 8 and 8.5). Fluorescence was recorded in a microplate reader (Perkin Elmer Envision) using 485/10 nm and 535/30 nm bandpass filters for excitation and emission, respectively. Raw fluorescence values were blank subtracted, normalized on neutral pH and plotted against pH values. In parallel, K+ sensitivity was tested by dissolving 10 μM mitoPOP in a solution containing 25 mM HEPES (pH 7) and 0, 150, or 300 mM KCl at 25°C. Raw fluorescence values (measured as described above) were blank subtracted, normalized on K+-free medium and plotted against [K+].The potassium probe mitoPOP was used to monitor the mitochondrial K4, 0.4 mM K2HPO4, 20 mM HEPES, 5.5 mM glucose, 1 mM CaCl2, pH = 7.4). Cells were washed once with Krebs-Ringer buffer and then incubated for 5 min in 140 mM Tris-KCl (pH 7.4). Thereafter increasing K+ or sucrose concentrations were added as indicated for the in situ response curve. Measurements were performed on a TriStar plate reader  at 25°C. Cells were subsequently counted by Trypan blue exclusion to normalize fluorescence readings to cell number. As a double control, to directly normalize the results to the amount of mitochondria independently of potentially variable cell numbers, cells plated in duplicates were analogously loaded with MitoTracker green  at 37°C for 30 min. The staining was washed once, the fluorescence intensity quantified as above in KCl buffer and used to normalize mitoPOP fluorescence values.Cells  were loaded with mitoPOP (1 μM) for 45 min at 37°C under cell culture conditions in modified Krebs-Ringer buffer  HeLa cells were seeded onto an Ibidi μ-slide  and transfected as previously described with pcDNA3 mtRFP and stained with mitoPOP for the last 45 min prior to visualization. The cells were visualized and images acquired on an LSM780 confocal microscope. Microscope, objective and aperture specifications may be found in Wilfinger et al. .p-values are indicated in the figure legends.All statistical analyses were performed using GraphPad  Prism v6 for Windows. Similarly, all graphs were generated with GraphPad Prism. One way ANOVA with Dunnett's posttest was performed in each case to assess significance. Individual + and Ca2+ release, we stably transfected HeLa cells with shRNAs targeted against LETM1, or randomly scrambled constructs. HeLa cell lines with LETM1 knockdown of varying efficiency were obtained. We observed that the most efficient viable knockdown had a 50% reduction in gene expression when compared to scramble controls (shSCR). This was achieved with shRNA LETM1 #2, while for another construct, shRNA LETM1 #1, no significant reduction was observed  or NCLX (SLC8B1), and found both genes had comparable transcription levels regardless of LETM1 expression  in our shLETM1 and control cells, and found no differences  of suspensions of control and LETM1 KD mitochondria  which accumulates in the mitochondrial matrix due to the latter's negative electrical potential . The specific localization of the probe was confirmed by co-localization with mtRFP in shSCR control cells   and 37°C . To additionally evaluate the role of the KHE in mitochondrial Ca2+ fluxes using a pharmacological approach, we resuspended shSCR cells in Na+-replete buffer, and quantified Na+-dependent Ca2+ fluxes in control cells in the absence or presence of the KHE inhibitor quinine. As shown in Figures +-dependent Ca2+ efflux in comparison to vehicle controls. These data support the hypothesis that the deregulation of mitochondrial monovalent cation homeostasis induced by KHE dysfunction reduces Ca2+ efflux.Using our shLETM1 cells we performed mitochondrial Can et al.   Figure . In the  Figures . Next, t Figures . For furTrypanosoma, C. elegans and Drosophila have demonstrated a role for this protein in K+/H+ exchange. While LETM1 has also been proposed to directly mediate mitochondrial Ca2+ uptake and efflux in Drosophila, mammalian cell culture and cell-free systems . A consequence of impaired KHE is K+ overload as has been demonstrated here and in yeast (Nowikovsky et al., Trypanosoma provide a substantial basis for the notion of an evolutionarily conserved function of LETM1 in KHE (Hashimi et al., In this study, we show that decreased LETM1 expression substantially reduced not only K Figures , which i Figures  and exte+ and Na+ exchange for protons. Further, we have demonstrated that LETM1-depleted mitochondria have a reduced Ca2+ efflux in buffer containing Na+, while the efflux is unchanged in a Na+-free buffer. Of note, the Na+-independent Ca2+ release only accounts for a minor part of the mitochondrial Ca2+ efflux. Although the data of Doonan et al. are both convincing and technically sound, little consideration is given to the key observation that there is no difference in Ca2+/H+ fluxes after the addition of CGP37157, an inhibitor of the mitochondrial NCLX. Careful consideration of these data illustrates that the proposed LETM1-mediated Ca2+/H+ fluxes are unchanged when LETM1 expression is reduced (Doonan et al., 2+/Na+ exchange. Contextually, this suggests LETM1 is not responsible for Ca2+/H+ fluxes in vivo. Furthermore, it has been reported that LETM1 does not mediate the bulk of electroneutral Ca2+ efflux in HeLa cells for which NCLX would instead be responsible (De Marchi et al., + concentration is expected to lower the activity of NCLX by decreasing the inward-directed Na+ chemical gradient. Furthermore, reduced monovalent cation translocation from the matrix would imply increased matrix alkalization, which might affect NCLX activity.We have shown that LETM1 depletion reduces mitochondrial K+ cycling and thereby indirectly modulates Ca2+ fluxes. Indeed, Na+-driven Ca2+ release was proposed to be regulated by the combined activity of the NHE with the NCLX to dissipate excess Na+ or with the HCHX to dissipate excess H+ (Carafoli, + and Na+ overload, the latter most likely slowing down the coordinated activities of NHE and NCLX, and thus resulting in decreased Ca2+ efflux. Less KHE activity most likely increases matrix alkalization, this also impinges on the NHE/NCLX. These simplified models, however, cannot account for the myriad of conditions, including changes of the matrix volume, increase of pH and the formation of Ca2+-phosphate precipitation, that play an important role in controlling the mitochondrial Ca2+ buffering capacity in vivo. Experimental systems using isolated cardiac mitochondria supported this notion (Blomeyer et al., 2+ buffering influence Ca2+ uptake and release, and thus Ca2+ sequestration, a non-negligible component of the mitochondrial Ca2+ cycle. Presumably, perturbations of matrix Na+ or pH also affect mitochondrial Ca2+ dynamics as highlighted earlier. Our results and model are in line with the idea of a mitochondrial Ca2+ cycle, which to maintain steady state, requires several components: MCU, NCLX, NHE, CHX, and KHE (Carafoli, + and likely matrix Na+. These perturbations significantly reduced mitochondrial Ca2+ efflux, reinforcing the importance of KHE activity for Ca2+ homeostasis. Despite our proposed mechanism of how LETM1 interplays with mitochondrial Ca2+ uptake and release via its role in mitochondrial monovalent cation homeostasis, the role of LETM1 EF hands in relation to osmotic regulation still needs to be addressed. Indeed, the presence of the EF hands has been reported to significantly affect Ca2+ uptake in mammalian systems (Doonan et al., 2+ uptake activity of MCU, one could speculate that this simple deletion elicits an unforeseen change in structure which prevents the activity or interaction with other crucial protein partners. However, these explanations are speculative and require further detailed investigation. Cell-free assay systems are indeed powerful tools to assess ion fluxes and protein activity. Nevertheless, these are simplified systems capable of reporting the activity of a single protein in isolation. Apparently, LETM1 can exhibit Ca2+/H+ exchange activity in vitro, as demonstrated by Tsai et al. or just recently by Shao et al. (Shao et al., +/H+ activities under physiologically relevant conditions are lacking. Whereas, experiments such as those by Hashimi et al. (2+/H+ or Ca2+/Na+ exchange.Based on the results presented above, we propose that LETM1 participates in Nai et al.  and De Mi et al.  continue+-mediated mitochondrial Ca2+ efflux is susceptible to changes of LETM1-dependent KHE and NHE activity. This confirms once again the central function of LETM1 in mitochondrial osmotic balance and offers explanations for the changes in Ca2+ dynamics observed in vitro. Indeed, by modifying the activity of the KHE and monovalent cation homeostasis, alterations of LETM1 function will affect mitochondrial Ca2+ cycling independently of the expression level of any Ca2+ cycle component.We believe our results may well conclude the debate on the fundamental function of LETM1. We have shown that NaSA and KN conceptualized and designed experiments. AM, DD, and MZ synthesized, validated and provided the mitoPOP probe. SA, MT, CP, and JS performed experiments, developed methods and analyzed data. SA, CP, and MT generated figures and performed statistical analyses. SA and KN wrote the manuscript. All authors approved the final version of the manuscript. KN conceived the project and supervised the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stromal-interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta that trigger store-operated Ca2+ entry (SOCE) via plasmalemmal Ca2+-selective Orai1 channels. While the STIM1 and Orai1 binding interfaces have been mapped, signaling mechanisms activating STIM1 recruitment of Orai1 and STIM1-Orai1 interaction remains enigmatic. Here, we show that ER Ca2+-store depletion rapidly induces STIM1 phosphorylation at Y361 via proline-rich kinase 2 (Pyk2) in ECs. Surprisingly, the phospho-defective STIM1-Y361F mutant formed puncta but failed to recruit Orai1, thereby preventing. SOCE Furthermore, studies in mouse lungs, expression of phosphodefective STIM1-Y361F mutant in ECs prevented the increase in vascular permeability induced by the thrombin receptor, protease activated receptor 1 (PAR1). Hence, Pyk2-dependent phosphorylation of STIM1 at Y361 is a critical phospho-switch enabling recruitment of Orai1 into STIM1 puncta leading to SOCE. Therefore, Y361 in STIM1 represents a novel target for limiting SOCE-associated vascular leak.Store-operated Ca We first stimulated ECs with thapsigargin in Ca2+ free media to induce Ca2+ release from ER (first peak) followed by repletion of 2 mM extracellular Ca2+ to activate SOCE (second peak). Overexpression of STIM1-WT or the phospho-defective STIM1-Y316F mutant had no effect on Ca2+ release or SOCE as compared to cells expressing control vector  is regulated by intracellular CaDepletion of Pyk2 also reduced SOCE  mimickinStudies show that upon store depletion, STIM1 appears in discrete puncta within the ER membrane that recruit Orai1 following translocation to ER-PM junctions2534To further corroborate the role of STIM1 phosphorylation on Y361 in inducing interaction with Orai1, we performed immunoprecipitation of Orai1 in HEK cells expressing either WT, phospho-defective (STIM1-Y361F), or phospho-mimetic (STIM1-Y361D) mutants. We found that thapsigargin induced the interaction of Orai1 with WT-STIM1 . However2+ release in cells transducing WT-STIM1 or control vector (data not shown) or phospho-defective STIM1-Y361F mutant . These findings thereby demonstrate a pivotal role of STIM1 phosphorylation on Y361 in mediating the lung vascular permeability response.We next addressed the physiological significance of STIM1 phosphorylation on its Y361 residue in regulating increased endothelial permeability. We first determined whether inhibition of STIM1 phosphorylation altered SOCE secondary to activation of the G-protein coupled receptor PAR1. Similar to thapsigargin, activation of PAR1 with thrombin also increased Pyk2 and STIM1 phosphorylation in a time dependent manner . Thrombi2+-selective Orai1 channel inducing SOCE. In this context, Pyk2-dependent phosphorylation of the Y361 residue within STIM1 induces SOCE. Our data showed that phosphorylation of the Y361 residue does not interfere with STIM1 puncta formation but controls recruitment of Orai1 to STIM1 puncta enabling STIM1-Orai1 interaction and Ca2+ entry through Orai1 channels. Furthermore, we established the physiological role of STIM1 tyrosine phosphorylation in regulating endothelial permeability in vivo by demonstrating that transduction of the phospho-defective STIM1-Y361F mutant in pulmonary endothelium of WT mice blocked the increase in endothelial permeability associated with activation of PAR1 signaling.In this study, we have uncovered a novel regulatory mechanism that primes STIM1 to interact with the Ca2+ in the ER lumen through its EF hand motif under basal conditions362+ depletion, which is followed by Ca2+ entry through SOCE2+ entry is a multistep process, which requires sensing of the Ca2+ concentration within the ER, establishment of ER- plasmalemmal (PM) junctions, and activation of plasmalemmal Ca2+ channels. STIM1 plays a critical role in each of these steps2+ concentration inside the ER lumen. Following agonist-induced depletion of the ER-Ca2+ store, STIM1 oligomerizes to form numerous puncta within the ER membrane2+ channels such as Orai1 channels to activate SOCEBoth STIM1 and Orai1 are integral components of SOCE in ECs212+ entry through Orai1 channels. We showed that Pyk2 but not p60Src is rapidly activated following stimulation of ECs with the SERCA inhibitor thapsigargin or thrombin. Depletion of Pyk2 inhibited both STIM1 phosphorylation and Ca2+ entry in ECs, demonstrating that Pyk2 mediates phosphorylation of STIM1. We showed that, while the expression of the phospho-defective STIM1-Y361F mutant markedly suppressed Ca2+ entry in ECs in response to either thapsigargin or thrombin, expression of the phospho-mimetic STIM1-Y361D mutant was sufficient to restore SOCE in Pyk2-depleted ECs, thus indicating that activation of Pyk2 through Ca2+ mobilization from ER stores provides a positive feedback mechanism for Ca2+ entry by phosphorylating STIM1.In the present study, we showed that Pyk2-dependent tyrosine phosphorylation of STIM1 at Y361 within SOAR domain of STIM1 is an integral step in activating Ca2+ depletion. This impairs recruitment of Orai1 into STIM1 puncta, limiting formation of functional Orai1 channels. Our findings also showed that recruitment of Orai1 into STIM1 puncta is a critical step in inducing Ca2+ entry via the SOCE, since expression of the STIM1-Y361F mutant or depletion of Orai1 in ECs inhibited SOCE to a similar degree.Our findings also showed that the phospho-defective STIM1-Y361F mutant exhibited a dominant negative effect through inactivation of endogenous STIM1. We speculate that the ectopically-expressed STIM1-Y361F mutant blocks SOCE by forming dysfunctional STIM1 oligomers with endogenous STIM1 protein upon ER-Caper se did not increase STIM1’s interaction with Orai1 basally indicating that phosphorylation of STIM1 alone is not sufficient to promote STIM1 and Orai1 interaction at the ER membrane and that consequently, STIM1 reorganization into puncta is a prerequisite for this interaction. We, therefore, conclude from these findings that recruitment of Orai1 into STIM1 puncta is an independent step requiring STIM1 phosphorylation by Pyk2.Crystal structure analysis revealed that the STIM1 dimer forms a V-shaped structure in which the Y361 residue from each monomer meet at the angle of the “V” shaped structureHow phosphorylation of Y361 regulates STIM1’s interaction with Orai1 remains to be parsed out but some speculation may be offered. Studies have identified SOAR as the minimal domain required for both STIM1 puncta formation and activation of Orai1 channels2534422+ entry and increased endothelial barrier permeability. This step is required for recruitment of Orai1 into STIM1 puncta and STIM1-Orai1 interaction. Our observations provide the first direct evidence for tyrosine phosphorylation-dependent activation of SOAR that mediates recruitment of Orai1 to STIM1 puncta. This phospho-switch is essential for activation of Ca2+ entry through Orai1 and disruption of endothelial barrier function in lungs, which leads to several pathologic disorders including lung injury. In this sense, STIM1 phsophorylation at Y361 represents a novel target for preventing diseases associated with leaky blood vessels.Activation of SOCE by STIM1 plays a key role in regulating endothelial permeability5556FUGENE transfection reagent was purchased from Promega . Primary antibodies against phospho-tyrosine , Orai1, Stim1, GFP as well as A/G agarose beads were purchased from Santa Cruz Biotechnology . Phospho-Pyk2 and pan Pyk2 antibodies were purchased from Cell Signaling Technologies . Thapsigargin was purchased from EMD Millipore . Paraformaldehyde was purchased from Fisher Scientific . Fura 2-AM was purchased from Life Technologies . Scrambled and Pyk2 siRNA were purchased from Ambion . All details are described in 2 and 95% air until they formed a confluent monolayer. Cells were detached using 0.05% Trypsin containing 0.02% EDTA and plated on 100 mm dishes. HEK cells were plated on 100 mm dishes and cultured using DMEM medium supplemented with 10% fetal bovine serum (FBS) till confluent.Human pulmonary arterial endothelial cells (HPAECs) and EBM-2 medium were purchased from . HPAECs were cultured in complete EBM-2 medium in a T-75 flask coated with 0.1% gelatin and supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified atmosphere of 5% COECs from the primary flask were detached using 0.05% Trypsin containing 0.02% EDTA. HPAEC were transfected with cDNA using FUGENE HD Transfection Reagent  or Amaxa electroporation (Lonza) as describedCells were lysed in radioimmunoprecipitation buffer  and were incubated with indicated antibodies overnight at 4 °C. After 24 h, lysates were coupled to protein A/G-agarose beads for 4 h at 4 °C. The beads were then collected by centrifugation at 3000 rpm for 3 min and washed three times using detergent-free radioimmunoprecipitation buffer. Phosphotyrosine of STIM1 was detected using a mixture of anti-PY20, anti-PY99 and anti-PY350 in equal proportion.2+ was measured using the Ca2+- sensitive fluorescent dye Fura 2-AM as described. Briefly, HPAE cells were transfected with indicated constructs for 24 h or siRNA for 48 h respectively. Cells were then loaded with Fura 2-AM dye for 20 min, rinsed twice with Ca2+ free HBSS media and stimulated with thapsigargin 2 μM or thrombin 50 nM to determine Ca2+ release. Only YFP transfected cells were selected for Ca2+ measurement. Ca2+ entry was determined following addition of 2 mM Ca2+. In each experiment, at least 5 YFP-tagged cells were imaged for Ca2+ analysis.An increase in intracellular CaCells seeded on 35 mm glass bottom dishes were co-transfected with Orai1-mCherry and YFP-STIM1 or YFP-STIM1-Y361F mutant using FUGENE. After 24 h, cells were serum starved for an hour in 0.1% FBS after which cells were treated with 2 μM thapsigargin for 5 min or left untreated. Cells were fixed with 4% formaldehyde and imaged using a LSM 880 inverted confocal microscope (Carl Zeiss) equipped with a Plan-Apochromat 63x/1.4 NA oil immersion objective, an Argon  and diode-pumped solid-state laser (λ = 561 nm), 2 photomultiplier tubes and Gallium arsenide phosphide detector. EYFP images were acquired with λ = 514-nm excitation and λ = 519–583 nm emission; mCherry images were collected with λ = 561 nm excitation and λ = 583–696 emission.2) of the cell.The 16-bit images were analyzed with Metamorph  and ImageJ (NIH) software. Projected images were generated by collecting maximum pixel intensity of the in-focus frames into a single frame. The images were threshold to remove background fluorescence and used to create a mask image. Using the mask images, number of Orai1-mCherry and YFP-STIM1 clusters was scored with automatic “Analyze Particles” algorithm of ImageJ software and using cluster size of 3–100 pixels and circularity 0.1–1.0. The data were expressed as the number of clusters per area  according to the manufacturer’s instructions. Threshold images were used to set the vertical and horizontal crosshairs to separate clusters into quadrants. The colocalization coefficient was calculated as the sum of colocalized pixels divided by the total number of Orai1-mCherry clusters. All images were prepared for publication using Adobe Photoshop.−/− mice were generated by crossing STIM1fl/fl with Tie2-cre58All animals used in this study were approved by the Institutional Animal Care and Use Committee of (IACUC) University of Illinois. EC-STIM1Liposomal delivery of cDNA into the mouse lung and lung vascular permeability measurements were performed in accordance with the IACUC. Cationic liposomes were made by mixing 9.5 ml of Chloroform and 315 μl of Dimethyl Dioctadecyl Ammonium Bromide (DDAB) with 200 μl cholesterol solution43STIM1−/− mice were anesthetized with an i.p. injection of ketamine (100 mg/kg) and xylazine (2.5 g/kg). PAR1 agonist peptide (TFLLRN-NH2) or control peptide (FTLLRN-NH2) (1 mg/kg body weight) were administered into mice through retroorbital route. After 30 min, left lungs were excised and completely dried in a 60 °C oven overnight for determining lung wet–dry ratio. All animals used in this study were approved by the Institutional Animal Care and Use Committee of (IACUC) University of Illinois.WT or EC-Generation of various constructs is described in t-test or Tukey test. Significance values are shown by *p < 0.05, **p < 0.01, and ***p < 0.001Graphs were generated using GraphPad Prism . Comparisons between experimental groups were made using one way ANOVA followed by How to cite this article: Yazbeck, P. et al. STIM1 Phosphorylation at Y361 Recruits Orai1 to STIM1 Puncta and Induces Ca2+ Entry. Sci. Rep.7, 42758; doi: 10.1038/srep42758 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


ATP plays important roles in coordinating the activities of astrocytes and neurons, and aberrant signalling is associated with neurodegenerative diseases. In rodents, ATP stimulates opening of Ca2+‐permeable channels formed by P2X receptor subunits in the plasma membrane. It is widely assumed, but not verified, that P2X receptors also evoke Ca2+ signals in human astrocytes. Here, we directly assess this hypothesis. We showed that cultured foetal cortical human astrocytes express mRNA for several P2X receptor subunits  and G protein‐coupled P2Y receptors . In these astrocytes, ATP stimulated Ca2+ release from intracellular stores through IP3 receptors and store‐operated Ca2+ entry. These responses were entirely mediated by P2Y1 and P2Y2 receptors. Agonists of P2X receptors did not evoke Ca2+ signals, and nor did ATP when Ca2+ release from intracellular stores and store‐operated Ca2+ entry were inhibited. We conclude that ATP‐evoked Ca2+ signals in cultured human foetal astrocytes are entirely mediated by P2Y1 and P2Y2 receptors, with no contribution from P2X receptors.Extracellular  Fibronectin was from Merck Millipore . Bovine serum albumin was from Europa Bioproducts Ltd . 2′‐amino‐UTP (2′‐amino‐2′‐deoxyuridine‐5′‐triphosphate) and 2′‐thio‐UTP (2′‐thio‐2′‐deoxyuridine‐5′‐triphosphate) were from Trilink Biotechnologies . Thapsigargin was from Bio Techne . qPCR primers and related reagents were from Qiagen . All other reagents, including 2‐aminoethoxydiphenyl borate (2‐APB), ADP, ATP, probenecid, UDP and UTP were from Sigma‐Aldrich . The properties of the drugs used are summarized in Table S1.Fura‐2 AM was from Invitrogen . Fluo‐8 AM was from Stratech Scientific . MRS2365 ((N)‐methanocarba‐2‐methylthio‐adenosine‐5′‐diphosphate), MRS2179  supplemented with 3% foetal bovine serum. Astrocyte growth medium includes human epidermal growth factor, insulin, ascorbic acid, gentamycin and l‐glutamine. Cells were passaged using trypsin, according to the supplier's instructions, when they reached 70–80% confluence. Cells were used for up to four passages after receipt, during which they maintained an astrocyte‐like morphology and expressed glial fibrillary acidic protein, assessed by qPCR and immunostaining.Human astrocytes isolated from foetal cortex were supplied as frozen cells that had not been passaged . The cells were confirmed, by Lonza, to be free of infection with HIV‐1 and hepatitis B and C, and we confirmed that they were free of mycoplasma. Astrocytes were grown at 37°C in humidified air containing 5% CO=E−CTPURINOCEPTORE−CTGAPDH, where E is the amplification efficiency, calculated as 10m, where m is the average increase in fluorescence for four cycles after the cycle threshold CT for the indicated PCR product. The effectiveness of all primer pairs was verified using BioBank generic pooled cDNA . All primers included in this study amplified a single product from the BioBank pooled cDNA , confluent cultures of astrocytes in 24‐well plates were lysed (200 μL cell processing buffer/well), mRNA was then isolated from the lysate (4 μL) and cDNA was synthesized using Fastlane cell cDNA kit . The cDNA was diluted fivefold with RNAase‐free water. Incubations for qPCR included Rotor‐Gene SYBR™ Green PCR master mix (10 μL), cDNA (5 μL), Quantitect primer assay  and RNAase‐free water (3 μL). In negative controls, the primers were omitted during qPCR or the reverse‐transcriptase was omitted during cDNA synthesis. A Rotor‐Gene 6000 thermocycler (Qiagen) was used for qPCR with a denaturation step , 40 amplification cycles  and then a melting curve (70–95°C). Expression of mRNA relative to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was calculated from: Relative expression A Figure . The melet al. 2, 1.5 mM CaCl2, 11.5 mM glucose and 11.6 mM HEPES, pH 7.3. After 60 min, the cells were washed with HBS, incubated for 90 min in HBS containing probenecid (2.5 mM) to allow de‐esterification of the indicator, washed and then used immediately for experiments. All experiments were performed at 20°C, to avoid extrusion and intracellular compartmentalization of fluo‐8, in HBS without probenecid. Where indicated, Ca2+‐free HBS containing BAPTA ethane‐N,N,N′,N′‐tetraacetic acid, final concentration 2.5 mM) was added immediately before stimulation to reduce the free [Ca2+] of HBS to < 100 nM.Confluent cultures of astrocytes grown in fibronectin‐coated 96‐well plates  were incubated at 20°C with fluo‐8 AM (4 μM) in HEPES‐buffered saline (HBS) containing probenecid (2.5 mM)  from: [Ca2+]i=KdF−FminFmax−F, where KD is the equilibrium dissociation constant of fluo‐8 for Ca2+ (389 nM), Fmin and Fmax are the minimal and maximal fluorescence values determined after addition of Triton X‐100 (0.2%) in Ca2+‐free HBS with BAPTA  or ionomycin (10 μM) in normal HBS (Fmax).Fluorescence  was recorded at 1.44‐s intervals using a FlexStation III fluorescence plate‐reader , which allows automated fluid additions during the recording  were loaded with fura‐2 by incubation with fura‐2 AM (2 μM) in HBS containing 2.5 mM probenecid . After a further 45 min in the same medium without fura‐2 AM, the cells were used for experiments at 20°C in HBS without probenecid. Imaging was performed using an Olympus IX71 inverted fluorescence microscope with alternating excitation (340 nm and 380 nm) provided by a Xe‐arc lamp at 1‐s intervals. Emission was recorded at 510 nm using a Luca EMCCD camera  and MetaFluor software . Background‐corrected ratios of F50 values  were determined. All analyses, except when otherwise stated, show pooled results from cells from two donors (Lonza lot numbers: 0000289765 and 0000402839). Key results from these and a third donor (0000514417) are shown individually in Figure For each experiment, the concentration‐response relationship was fitted to a logistic equation  from which the maximal amplitude and pEC2+]i  in populations of cultured foetal human cortical astrocytes. Similar results and with similar sensitivities to ATP, but with Ca2+ signals of different amplitude, were observed in cells from three different donors , but the sustained phase of the response was abolished  and three of the seven subunits of P2X receptors   to quantify expression of these mRNAs in cultured foetal human cortical astrocytes. The results confirmed expression of mRNA for four of the eight subtypes of P2Y receptors (P2YX6) Fig. .2+ signals in human astrocytes. This contrasts with the prominent role ascribed to P2X receptors in rodent astrocytes. We therefore assessed whether P2X receptors contribute to the Ca2+ signals evoked by ATP in human astrocytes.Since some P2Y receptors, but no P2X receptors, can stimulate PLC  evoked by α,β‐meATP and BzATP were 5 ± 0 nM and 5 ± 3 nM respectively (n = 3); the parallel measurements of ATP‐evoked Δ[Ca2+]i were 132 ± 26 nM and 150 ± 16 nM. We avoided higher concentrations of α,β‐meATP (100 μM) and BzATP (300 μM) because they evoked Ca2+ signals in Ca2+‐free HBS (not shown).We detected mRNA for P2Xtes Fig. e. P2X4 a2+ signals evoked by P2Y receptors to unmask any possible contribution from P2X receptors. This required inhibition of both the Ca2+ release and Ca2+ entry components of the response evoked by P2Y receptors   had no significant effect on [Ca2+]i  to thapsigargin‐treated cells in normal HBS had no significant effect on [Ca2+]i  receptors, there was the expected increase in [Ca2+]i, but subsequent addition of α,β‐meATP to stimulate P2X receptors (30 μM after 5 min) evoked no further increase in [Ca2+]i  are coupled to Gq/11 and can thereby stimulate PLC. We used ligands that distinguish between the subtypes for which mRNA was detected to resolve the contributions of different P2Y receptors to the ATP‐evoked Ca2+ signals (Table S1).All four of the P2Y receptor subtypes for which mRNA was detected in human astrocytes  . MRS2365 evoked a concentration‐dependent increase in [Ca2+]i  and the maximal amplitude of the response was similar to that evoked by ADP . UDP had no effect on [Ca2+]i   and 2′‐thio‐UTP  , the ΔpEC50 value for MRS2365 was 0.64 ± 0.26   caused a smaller shift in the ΔpEC50 value for the Ca2+ signals evoked by ATP  . We conclude that P2Y1 and P2Y2 receptors, but not P2Y6 or P2Y11 receptors, evoke Ca2+ signals in cultured foetal human astrocytes.UTP is a potent agonist of P2Y 5) Fig. c. The efi Figure B. Hence,) Figure A. In kee) Figure B. The sh1 receptors (with ADP) and of P2Y2 receptors (with UTP) are entirely responsible for the Ca2+ signals evoked by ATP, we compared the maximal amplitudes of the responses evoked by the three stimuli in parallel measurements. The Δ[Ca2+]i evoked by ATP, ADP and UTP were 142 ± 5 nM, 88 ± 11 nM and 51 ± 13 nM (n = 3) respectively. Hence, the sum of the responses to ADP and UTP (139 ± 25 nM) was not significantly different from the response evoked by ATP (142 ± 5 nM). These results confirm that ATP evokes Ca2+ signals through P2Y1 and P2Y2 receptors, but they do not resolve whether the two receptors are expressed in different cells or whether both contribute to the responses in individual cells. We therefore examined the responses of single fura‐2‐loaded cells to ATP, UTP and ADP.To determine whether activation of P2Y1 and P2Y2 receptors. Analyses of responses to sequential stimulation with ADP and UTP revealed that 59 ± 8% of the cells that responded to ADP then responded to UTP , while 92 ± 6% of cells that responded to UTP responded to a subsequent challenge with ADP , suggesting that about half of the cells responded to both stimuli. These results demonstrate that most cells respond to ATP and that many express both P2Y1 and P2Y2 receptors. We considered whether autocrine release of ATP might contribute to the sustained phase of the Ca2+ signal evoked by selective activation of P2Y receptors. This seems unlikely, since in cell populations the relative amplitudes of the initial and sustained phases were similar for cells stimulated with ATP to activate all P2Y receptors or with ADP to activate only P2Y1 receptors  or P2Y2 receptors (228 cells) responded during the next 3–6 min with a detectable increase in [Ca2+]i.In these single‐cell analyses, 87 ± 6% of cells responded to ATP , 65 ± 5% responded to ADP  and 41 ± 6% responded to UTP , suggesting that at least 22% of ATP‐responsive cells express both P2Y2+ signals. We showed that mRNAs for four P2Y receptors (P2Y6 ~ P2Y11 > P2Y2 > P2Y1) and three P2X receptor subunits (P2X6 > P2X4 > P2X5) are expressed. There was no detectable mRNA for any of the remaining P2 receptors  did not increase [Ca2+]i   were not associated with expression of functional P2Y receptors. In rodents too, there is no functional response to P2Y6 receptors, although their mRNA is expressed (Fumagalli et al. 2+ signals (Verkhratsky et al. Our analyses of mRNA for P2X receptors were not predictive for expression of functional plasma membrane receptors. Others have also noted expression of mRNA for P2 receptors for which there was no corresponding functional response (Fumagalli 2+ signals that are entirely mediated by P2Y1 and P2Y2 receptors, each of which stimulates PLC and thereby IP3‐evoked Ca2+ release and SOCE. Many astrocytes express both of these receptors, but some express only one or the other. We have not further explored this heterogeneity. Although mRNA for P2X receptor subunits is expressed, P2X receptors do not contribute to ATP‐evoked Ca2+ signals.We conclude that in cultured foetal cortical human astrocytes, ATP evokes CaFigure S1. Melting curves for qPCR analyses of the expression of purinoreceptor subtypes.Figure S2. ATP evokes Ca2+ signals through P2Y receptors in astrocytes from three donors.Figure S3. Stimulation of P2Y receptors does not cause translocation of functional P2X receptors to the plasma membrane.Figure S4. Neither P2Y6 nor P2Y11 receptors evoke Ca2+ signals in cultured human foetal astrocytes.Figure S5. 2′‐azido‐UTP does not evoke Ca2+ signals.Figure S6. Effects of MRS2179, a selective antagonist of P2Y1 receptors, on the Ca2+ signals evoked by ATP and MRS2365.Table S1. Properties of the drugs used.Table S2. Primers used for qPCR analyses.Table S3. Ca2+ signals evoked by P2Y‐selective agonists in cultured human foetal astrocytes.Click here for additional data file.


Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; however, the specific rates are highly dependent on the stage of the disease upon diagnosis. Current screening and imaging tools are insufficient to detect early lesions and are not capable of differentiating the subtypes of ovarian cancer that may benefit from specific treatments.As an alternative to current screening and imaging tools, we utilized wavelength dependent collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe the structural differences in the extracellular matrix (ECM) of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous tumors.The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size scale and encode structural information. The wavelength dependence of these readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found a strong wavelength dependence of these metrics that are related to significant structural differences in the collagen organization and are consistent with the dualistic classification of type I and II serous tumors. Moreover, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. The SHG metrics and optical scattering measurements were used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (>90%) between high-grade serous tumors from the other tissue types. High-grade serous tumors account for ~70% of ovarian cancers, and this delineation has potential clinical applications in terms of supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool.SHG and optical scattering measurements provide sub-resolution information and when combined provide superior diagnostic power over clinical imaging modalities. Additionally the measurements are able to delineate the different subtypes of ovarian cancer and may potentially assist in treatment protocols. Understanding the altered collagen assembly can supplement histological analysis and provide new insight into the etiology. These methods could become an in vivo screening tool for earlier detection which is important since ovarian malignancies can metastasize while undetectable by current clinical imaging resolution. Ovarian cancer remains the most deadly gynecological cancer among women with an aggregate 5-year survival rate of ~45%. However, the specific rates are highly dependent on the stage of the disease upon diagnosis. For example, diseased localized to the ovary has a 5-year survival rate of ~92%, whereas this decreases precipitously to 27% for metastatic disease. With current methods only 15% percent of the patients are diagnosed at stage I . Early detection is difficult due to vague symptoms  as well as the lack of effective clinical screening/imaging tests. Although the CA125 tumor marker and trans vaginal ultrasound (TVUS) have been investigated as screening strategies, both of these methods are not sufficiently selective or specific to be employed as clinical diagnostic tests for early detection of ovarian cancer .Recently, genetic analysis has led to identification of several subtypes of ovarian tumors , 5. TypeExamination of remodeling of the extracellular matrix (ECM) through higher resolution and highly specific approaches holds great promise in helping to address these diagnostic needs. This is because these alterations are thought to be a critical step in the initiation and progression of many epithelial carcinomas . These aP = χ(2)EE where P is the induced polarization, E is the electric field vector of the laser, and χ(2) is the second-order nonlinear susceptibility tensor. The nonlinear susceptibility tensor χ(2) dictates the intensity of the SHG signal and requires a non-centrosymmetric assembly of harmonophores, which have permanent dipole moments on the size scale of λSHG, to be nonvanishing. This technique is collagen specific and permits imaging deep into tissues (few hundred microns) with intrinsic optical sectioning [To investigate this possibility, we implemented high resolution optical microscopy and spectroscopy tools to quantify changes in the ECM across a spectrum of human ovarian cancers. We use Second Harmonic Generation (SHG) imaging microscopy  to objecctioning . The undctioning .k = kω2 − 2kω = 0, . Biological phasematching conditions are not ideal (i.e. Δk ≠ 0), and as a result of conservation of momentum, there is a specific emission pattern (i.e.forward (FSHG) and backward (BSHG) components) that depends on the tissue structure. We thus utilize the intensity ratio, FSHG/BSHG, as a metric that arises from the fibril size and packing relative to the SHG wavelength, λSHG. Phasematching also has implications on the observed SHG intensity, where the relative SHG intensity scales as sin(mΔkL/2) where m is an integer and thus becomes less efficent for larger phase mismatch, i.e. larger Δk values, which correspond to more random structures compared to the length scale of λSHG [SHG/BSHG values, that based on a mathematical model we developed [SHG microscopy has been used in previous studies to investigate the alterations of the stroma in human and mouse models in ovarian cancer using image analysis approaches that interrogated the fiber alignment –15. We p of λSHG . For noreveloped , were coeveloped . These developed , 19.We now extend those earlier efforts and perform these measurements and analysis for several tumor types  and also across a large excitation wavelength range (780–1160 nm). We will show the wavelength dependencies encode structural information that identifies changes in collagen fibril/fiber assemblies. All the SHG and optical scattering metrics are reflective of the size and distribution of ECM components relative to the interrogating wavelength. These studies on ECM alterations will lead to better insight into ovarian cancer etiology and progression.en face using a Leica Vibratome 1200S  to thickness of 50 μm and 100–150 μm for optical scattering measurements and SHG imaging studies, respectively. Tissues were classified by a gynecological pathologist into normal (n = 4), benign (n = 4), borderline/low-grade serous Type I (n = 4), endometrioid Type I (n = 3), and high-grade serous Type II (n = 3). The genomic mutations of the tissues are not known.Malignant ovarian tissues were obtained using an IRB approved protocol from consented patients undergoing surgical de-bulking treatment for ovarian cancer. Normal ovaries were obtained from consented patients (aged 45–65) undergoing bilateral salpingo-oophorectomy for benign neoplasms, fibroids, endometriosis, and uterine prolapse. All tissues were immediately fixed in 4% formalin and refrigerated for 24 h and then switched to phosphate buffered saline (PBS). The specimens were sectioned http://loci.wisc.edu/software/wiscscan) on a fixed stage upright microscope base  microscope. A 40 × 0.8 numerical aperture (NA) water immersion objective and a 0.9 NA condenser are used for excitation and collection, respectively, of the forward SHG signal providing lateral and axial resolutions of approximately 0.7 and 2.5 μm, respectively. The backward SHG was collected in a non-descanned geometry, where the detector was in the infinity space. Both detectors are H7422-40P GaAsP photomultiplier tubes . Calibration of the forward and backward detection pathways was performed using the two-photon excited fluorescence imaging of beads emitting in the same wavelength range as the collected SHG signal. The laser excitation range was 780–1160 nm provided by Chameleon Ultra Ti:Sapphire oscillator and a synchronously pumped APE Optical Parametric Oscillator . For all excitations, the SHG signal was isolated with 20 nm full width half maximum bandpass filters centered at the corresponding SHG wavelength . Circularly polarized light, determined at the focus, was used throughout to equally excite all fiber orientations [The essentials of the SHG imaging system has been described elsewhere . A femtontations .Depth dependent Forward/Backward (F/B) measurements were made for the entire thickness of the ovarian sections in three different locations, from which the data was obtained sequentially across the wavelength range. The SHG intensities were integrated across the whole fields of view using both FIJI   and MATLSHG/BSHG, and relative SHG conversion efficiency were decoupled from the depth dependent SHG response curves using Monte Carlo simulations based on an adapted MCML (Monte Carlo Multi-Layer) [SHG/BSHG at each wavelength was extracted by running a series of forward simulations of the measured forward/backward vs depth curve based on the corresponding measured optical properties (described below) and initial guesses of the emission directionality and then obtaining the best fit to the simulations. The relative SHG conversion efficiency at each excitation wavelength was determined by modeling the normalized forward SHG attenuation response using a similar simulation incorporating the extracted FSHG/BSHG and wavelength specific optical parameters at both the excitation and SHG wavelengths. All forward simulations were completed using parallel computing at the Wisconsin Center for High Throughput Computing at the University of Wisconsin-Madison and the values were stored in tables, permitting all the image processing to be performed in custom MATLAB scripts.The SHG directional emission ratio, Fi-Layer)  framewori-Layer) , 23. Then, absorption coefficient μa, scattering coefficient μs, and scattering anisotropy g. An estimate of the bulk refractive index was determined by a total internal reflection measurement of the sample as described by Li et al. [μa < < μs and was previously confirmed in ovarian tissues [We determined the optical properties of tissue through an average of three independent locations within a tissue specimen, where these parameters include the refractive index i et al. . The abs tissues .μa < < μs [μs from the Beer-Lambert law. We then report the reduced scattering coefficient, μs′, which is a merged property defined by:The scattering coefficient and anisotropy were obtained independently in a multi-step process using on-axis attenuation measurements which is applicable when  < < μs . These vSHG/BSHG, μs, and their wavelength dependence was used to classify the images. ROC curves were computed by comparing the modeled response versus the true or assigned tissue classification via a logistic regression. All the ANOVA and Fisher LSD statistical tests were performed in Origin 9.1 . For the latter, p < 0.05 was considered significant.The canonical variable linear discriminant and logistic progression were performed in SAS  using the CANDISC procedure, where this was applied to all sample data points. Using the canonical variable weights, a model based on the relative SHG conversion efficiency, FThe left and right columns of Fig. It is difficult to precisely determine fiber diameters as they are not all discrete and often overlap within the resolution of the microscope. However, we approximated the diameters by thresholding and measuring the thickness by creating a profile across several discretized fibers. We then found average values (standard error in parenthesis) of: normal: 2.7 (0.1) μm; benign: 3.6 (0.2) μm; endometrioid: 1.9 (0.1) μm; LGS: 3.4 (0.2) μm and HGS: 2.3 (0.1) μm. The diameters of the LGS and benign were similar; those of the endometrioid and HGS were similar, but all the others were different from each other.μs′, depends on the spatial distribution of the refractive index due to structural differences on size scales smaller than the diffraction limit. For rigorous tissue characterization, Backman et al. used the flexible Whittle-Matérn correlation function to quantify the distribution of length scales where the output is the shape factor m, which corresponds to one half of the fractal dimension [s′(λ) ~ λ(2m-4) where higher m values are associated with larger, more ordered structures on the approximate size scale of 50 nm to 1 μm.The wavelength dependence of the reduced scattering coefficient, imension , 27. Theμs′, for the five tissue types over the range of 390–535 nm (corresponding to the wavelengths where the SHG metrics were probed) and the results are shown in Fig. μs′ values reflect both the fiber and cellular components, however the former is much more strongly scattering. The other tissues  showed intermediate behavior. These were mostly statistically different at 390 nm (p < 0.05), and some were significant at other wavelengths. In addition to the absolute scattering intensities, we also compared the tissues through the fitted m values. By this metric, LGS and endometrioid tissues are the most ordered with m values of 1.41 and 1.40, respectively, where these were not distinct; however, the μs′ were different at 390 and 445 nm. Benign tissues (m = 1.01) show the least order, followed by HGS (m = 1.17), then normal tissues (m = 1.32). Given that both μs′ and m are different for some of the tissues, we concluded that both the scattering intensity and spectral dependence are needed to describe the tissues fully and provide the best differentiation. We note that this is not unexpected as the m values correspond to organization on the ~50 nm-1 μm size scale, while μs′ is a merged parameter reflecting both organization (g) and density (μs). Thus, higher values of both parameters may not occur for a single tissue.We measured the spectral dependence of the reduced scattering coefficient, SHG/BSHG metric is a subresolution parameter that arises from the fibril size and packing relative to λSHG [SHG/BSHG. Here we utilize a broad excitation range (780–1160 nm), which is limited by optical transmission of the SHG (390 nm) and excitation (1160 nm) wavelengths and then extract FSHG/BSHG as described in the The F to λSHG . The wav(2) test) from the measured data, implying a good fit to FSHG/BSHG. All ovarian tissue types displayed an increase in FSHG/BSHG emission directionality with increasing wavelength. This result is expected due to phasematching considerations, as we have reported for other tissues [The data are summarized in Fig.  tissues . This ar tissues .Fig. 3ExSHG/BSHG is tissue specific. HGS tumors have the smallest FSHG/BSHG. One physical scenario that gives rise to a low FSHG/BSHG is when the fibril size and spacing are regular and much smaller than λSHG where this results in efficient quasi-phasematching (QPM), thus increasing the BSHG emission [SHG/BSHG emission directionality indicating this tissue type has the largest collagen fibrils. This is also suggested by this tissue having the largest measured fibers from Fig. SHG/BSHG values between HGS and LGS tissues, demonstrating that both these tissues have altered collagen assembly than normal post-menopausal ovarian stroma. However, the alterations result in opposite trends, suggesting the modifications have different forms. This is consistent with type I and type II classes representing genetically different diseases [However, the form of the wavelength dependent increase in Femission , 28. We emission . The higdiseases .SHG/BSHG metric was 988 nm. The FSHG/BSHG values and statistical analysis at this wavelength are shown in Fig. SHG/BSHG statistically differentiates the collagen fibril size and assembly of HGS tissues from LGS tissues and also from benign tissues (p ≤ 0.002) despite small samples sizes of 3 or 4 patients per tissue type. Benign tissues are also statistically different (p ≤ 0.01) from normal and endometrioid tissues. Also interesting is the difference in collagen fibril assembly of LGS and the endometrioid tissues (p = 0.01) highlighting the heterogeneity of collagen features within these tissues, even though both are classified as Type I [The optimal single wavelength to differentiate the tissue types by the Fs Type I . AlthougSHG [SHG/BSHG values.We also determined the wavelength dependence of the relative SHG conversion efficiency as a means to characterize the tissue structure. The conversion efficiency is a coupled effect of the collagen concentration (square thereof) and its fibrillar organization on the size scale of λSHG , 22. Monp ≤ 0.01) from normal and all other types of ovarian cancer. LGS, endometrioid, benign, and normal tissues had similar values of relative SHG conversion efficiency; however, the wavelength dependent response is slightly different. LGS tissues have the greatest wavelength dependent increase in the SHG conversion. We note that the HGS conversion efficiency decreases with wavelength, whereas the other tissues show an increase, suggesting large structural differences which will be addressed in the Discussion.The extracted SHG conversion efficiencies for the five tissue types across the 780–1070 nm range are shown in Fig. In this study, three sub-optical resolution properties were obtained to further understand the ECM remodeling associated with ovarian cancer. Combining these independent metrics provides a more robust characterization of the normal and diseased ovarian stroma. This is important as not all parameters at all wavelengths were statistically different. To this end, we used a linear discriminant with three canonical variables see , where tSHG/BSHG emission directionality [SHG produce larger FSHG/BSHG, where smaller sizes yield lower values, and also weaker SHG intensities. A special case can arise, when the fibrils are much smaller than λSHG and equally spaced, through QPM, producing relatively more efficient backward SHG.We previously developed a heuristic model based on phasematching between the excitation and SHG waves that correlates collagen fibril size and assembly with the Fionality . In thisSHG; thus, if the tissues have different distributions of domain sizes, this will result in a different wavelength dependence of the FSHG/BSHG parameter. The overall increase in FSHG/BSHG with increasing wavelength is expected by simple phasematching conditions based on the wavelength dependence of the refractive index [SHG. The HGS tissues show the weakest wavelength dependence, and this is consistent with our previously reported TEM images, which showed the fibrils were all ~60 nm in diameter [SHG. The low FSHG/BSHG is further consistent with arising from the QPM mechanism. While we do not have TEM images for the LGS and benign tissues, within the resolution of the microscope  which is a property of the collagen itself, and phasematching, which arises from the organization relative to λSHG [(2) rather than the improved phasematching at longer wavelengths [SHG/BSHG values. Here, we performed this analysis on the different ovarian ECM tissues and compared their relative conversion efficiencies and wavelength dependence.Differences in collagen density and organization are also manifested in the relative SHG conversion efficiencies. The SHG conversion efficiency is based both on the magnitude of nonlinear susceptibility tensor χ to λSHG . We receelengths . Furtherelengths , and as (2) component dominates the increased phasematching at longer wavelengths. This is further consistent with the lower FSHG/BSHG emission directionality shown in Fig. SHG with a narrow distribution of diameters. The relative SHG conversion efficiencies were similar at 988 nm . All the other tissue types showed a slight increase with wavelength. From a structural perspective, this implies the fibril domain is larger than the HGS tissues and is also characterized by a larger distribution of fibril sizes  were largely similar. In analogy to the SHG data, the scattering coefficients are also related to the scatterer size relative to the interrogating wavelength. The higher sensitivity at the shorter wavelengths implies that the scattering structures lie within this size range. Although optical scattering and SHG arise from different physics, we found similar size scales are operative in both cases. The data identified the most sensitive wavelength range to use for diagnostic purposes for both SHG and optical scattering.We also characterized the tissue architecture using optical scattering measurements of both the scattering intensity and its wavelength dependence Fig.  18, 19,,19, 26. SHG/BSHG emission directionality, relative SHG conversion efficiency, μs′, and their respective wavelength dependence provided differentiation in some cases, each metric described only a single feature of the ovarian ECM. Combination of the metrics in a canonical linear discriminant provided enhanced classification based on the weights of the three individual variables. Robust classification (>90%) of high grade serous from all other ovarian tissue types was achieved. This is arguably the most important task as these tumors account for 70% of all ovarian malignancies and have the poorest survival rates. Moderately accurate classification was obtained for normal tissues relative to the other types. Interestingly, the least accuracy was obtained for LGS and benign tumors  increased accuracy of clinical classification without the need of genomic analysis; ii) improve our understanding of the different etiologies of different ovarian cancers; and iii) potential to develop ovarian cancer type specific chemotherapeutics. This work provides the core data to show the potential of ECM collagen measurements as a new image-based biomarker for ovarian cancer. Future efforts will include further examination of collagen associated metrics, clinical trials to further show the linkage between collagen changes and clinical outcome, and the design of instrumentation that can exploit this information for clinical use.


It acts as a cofactor of several important enzymes, as a regulator of ion channels such as voltage-dependent Ca2+ channels and K+ channels and on Ca2+-binding proteins. In general, Mg2+ is considered as the main intracellular antagonist of Ca2+, which is an essential secondary messenger initiating or regulating a great number of cellular functions. This review examines the effects of Mg2+ on mitochondrial functions with a particular focus on energy metabolism, mitochondrial Ca2+ handling, and apoptosis.Mg Similar by Mg2+ . However by Mg2+ . Resultsnium red . Finallynium red  has sugg2+ does not affect the rapid mode of mitochondrial Ca2+ uptake [2+ transport through the IMM distinct from MCU [Recent study documented that Mg+ uptake  that repfrom MCU .2+ release has previously been demonstrated to depend on the Ca2+-induced release of Ca2+ from mitochondria (mCICR). The mechanism of mCICR involves the transitory opening of the PTP operating in a low conductance mode. Therefore, Ca2+ fluxes from mitochondria are a direct consequence of the mitochondrial depolarisation spike (mDPS) caused by PTP opening [2+ waves. Mitochondria therefore appear to be excitable organelles capable of generating and conveying electrical and Ca2+ signals. In living cells, mDPS/mCICR is triggered by IP3-induced Ca2+ mobilisation leading to amplification of the Ca2+ signals primarily emitted from the ER [2+ concentration [2+ on Ca2+-dependent opening of PTP [The main route of mitochondrial Ca opening . In vitrm the ER . As docuntration . This isg of PTP .2+ release/efflux [+/Ca2+ exchanger, whereas in excitable tissues , it appears to be primarily a Na+/Ca2+ exchanger [+/Ca2+ exchange was identified in 2010 [+/Ca2+ exchange (Letm1) was reported in 2009 [+/Ca2+ exchanger mediating Ca2+ extrusion from mitochondria. The transport activity of the Na+/Ca2+ exchanger is inhibited by Mg2+ at concentration 2.5 mM [2+ does not inhibit the Ca2+ flux mediated by the H+/Ca2+ exchanger Letm1, even at ∼300-fold excess [Two additional antiporters are suggested to play an important role with respect to mitochondrial Cae/efflux , 57. In xchanger , 72. The in 2010 . A possi in 2009 , althoug in 2009 , 76. As  in 2009 , furthern 2.5 mM , whereasd excess .Mitochondria play an important role in the process of the intrinsic pathway of apoptosis , 79. The2+ in apoptosis [2+ has been largely ignored. Several in vitro studies have suggested the stimulatory role of Mg2+ in both the extrinsic and intrinsic pathways of apoptosis. Changes in cytosolic Mg2+ concentration have been observed in the glycodeoxycholate-induced apoptosis of hepatocytes [2+ observed in early phase of apoptosis has been explained by Mg2+ being necessary to stimulate the activity of Ca2+/Mg2+-dependent endonucleases, which are the executors of apoptosis. Patel et al. [2+-free medium prevents the rise in intracellular Mg2+ and reduces nuclear DNA fragmentation. On the contrary, Chien and coworkers [2+ is independent of the extracellular Mg2+ concentration and the source of the elevated intracellular Mg2+ has been suggested to be in the mitochondria. This suggestion is supported by the discovery of mitochondrial Mg2+ efflux and influx transporters [2+ from mitochondria with preserved integrity  as the response to the apoptotic compound, gliotoxin [2+ concentrations in some models of the induction of apoptosis.In contrast to the well-established role of Capoptosis , the rolatocytes , during atocytes  and in tatocytes . The elel et al.  have shooworkers  have docsporters , 16 and liotoxin . Finallyliotoxin . On the liotoxin  and the liotoxin  Mg2+ con2+ on cytochrome c release from mitochondria, an event that is followed by apoptosome formation and further progression of mitochondrial apoptosis [2+ has been suggested, the impact of Mg2+ on cytochrome c release seems to depend on the mechanism of OMM permeability increase. The release of both Bax- [2+. On the contrary, Noxa-induced cytochrome c release is inhibited by Mg2+; this can be explained by the ability of Mg2+ to inhibit PTP [Previous studies have also documented the impact of Mgpoptosis . Althougoth Bax-  and tBidoth Bax-  has beenibit PTP , since Pibit PTP .2+ affects mitochondrial functions that have an important impact on cell survival. Recent work on Mrs2 knockdown HeLa cells has unambiguously revealed that the disruption of mitochondrial Mg2+ homeostasis has a dramatic impact on a cellular energy status and cell vulnerability [2+ balance [2+ and mitochondria. Since mitochondria display significant cell and tissue heterogeneity [2+ on cellular physiology can also be anticipated to be cell- and tissue-type-dependent. Experiments on a variety of cell types will be important. In addition, the impact of Mg2+ on apoptosis initiation and execution in various cells has to be investigated in more detail. With respect to apoptosis, the cell-type specificity and the cause-consequence relations between apoptosis initiation and changes in the intracellular or mitochondrial concentration of Mg2+ are still unclear. Moreover, recent studies strongly point to the importance of ER-mitochondria interactions with respect to mitochondrial functions, Ca2+ homeostasis, and dynamics [2+ is not as clear yet, the study of the transport of Mg2+ through the ER membrane and the possible impact of the luminal Mg2+ concentration on ER-mitochondria crosstalk and on mitochondrial Mg2+ transport and functions will be crucial. Finally, other processes are localised in the mitochondria, which are also considered as the main site of the intracellular production of reactive oxygen species. The effect of Mg2+ on these processes has not been discussed in this review, but some interest should be focused on this direction in the future.Mitochondrial dysfunction has been implicated in the mechanisms of several serious human pathologies including metabolic , 94, carrability . Moreove balance . These fogeneity , 99, thedynamics , 101. Si


Idelalisib, a PI3K inhibitor, specifically targeting p110δ, has been approved for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma and follicular lymphoma. However, the mechanisms of action of idelalisib in colon cancer cells are not well understood. We investigated how idelalisib suppresses colon cancer cells growth and potentiates effects of other chemotherapeutic drugs. In this study, we found that idelalisib treatment induces PUMA in colon cancer cells irrespective of p53 status through the p65 pathway following AKT inhibition and glycogen synthase kinase 3β (GSK3β) activation. PUMA is necessary for idelalisib-induced apoptosis in colon cancer cells. Idelalisib also synergized with 5-FU or regorafenib to induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed apoptosis and antitumor effect of idelalisib in xenograft model. These results demonstrate a critical role of PUMA in mediating the anticancer effects of idelalisib in colon cancer cells and suggest that PUMA induction can be used as an indicator of idelalisib sensitivity, and also have important implications for it clinical applications. Idelalisib, also known as CAL-101 and GS-1101, is the first-in class phosphatidylinositol 3 kinase delta (PI3Kδ) inhibitor, a cytoplasmic tyrosine kinase involved in a number of signaling pathways within B-cells –4. HowevPUMA, p53 upregulated modulator of apoptosis, belongs to BH3-only Bcl-2 family, which play a key role in apoptosis in cancer cells , 7. PUMAOur results demonstrated that PUMA induction by idelalisib via the AKT/GSK-3β/NF-κB pathway and play a pivotal role in therapeutic response to idelalisib in CRC. These results indicated that PUMA induction is indicative of the therapeutic efficacy of idelalisib, and likely other targeted agents as well.p53-KD) HCT116 cells is similar  HCT116 cells. Idelalisib treatment induced significantly apoptosis in HCT116 cells, which was significantly reduced in PUMA-KD cells , which leading to GSK3β inactivation in both cell lines [Next, we determined if GSK3β is involved in idelalisib-induced p65 activation. First, we found that GSK3β siRNA but not the control siRNA suppressed idelalisib-induced nuclear translocation of p65 Figure . GSK3β dll lines   is the most common malignancy with the third largest incidence and mortality among all diagnosed cancers in the worldwide . About 2In the present study, idelalisib induces PUMA expression through GSK-3β/NF-κB pathway following AKT inhibition and initiates apoptosis through the intrinsic apoptosis pathway in colon cancer cells. PUMA induction plays a key role in apoptosis in response to varieties chemotherapeutic agents, and is likely to be a useful indicator of chemo sensitivity. Previously reports showed that PUMA induction matches to differential sensitivity to EGFR TKIs, and loss of PUMA induction is associated with insensitive to EGFR TKIs , 32. Furp53-independent pathway. Idelalisib-induced PUMA expression change may functions as a biomarker for its clinical trials, and can help important implications for the future development and application.In conclusion, our results demonstrated a novel antitumor mechanism of idelalisib through PUMA-mediated apoptosis in a The human colon cancer cell lines, HCT116, DLD1, HT29, Lim2405, and LoVo were got from American Type Culture Collection . All colon cancer cell lines were cultured in DMEM medium supplemented with 10% heat-inactivated newborn calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). NCM356 was got from INCELL , and cultured in M3 media according the supplier's instructions. The anticancer agents and chemicals used include idelalisib, regorafenib (Selleckchem), BAY 11–7082 (Merck), 5-fluoreuracil  were diluted with DMSO. Constitutively active AKT was got from addgene [4 cells/well. After overnight incubation, various concentrations of idelalisib were added into wells and incubated for additional 72 hr. 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed using the MTS assay kit (Promega) according to the manufacturer's instructions. Luminescence was measured with a Wallac Victor 1420 Multilabel Counter (Perkin Elmer). Each assay was conducted in triplicate and repeated three times.Indicated cell lines were seeded in 96-well plates at a density of 1×10TM Probes Supermix (Bio-Rad) in a final reaction volume of 20 μL with gene-specific primer/probe sets, and a standard thermal cycling procedure (35 cycles) on a Bio-Rad CFX96TM Real-time PCR System. PUMA and β-actin levels were assessed using TaqMan Gene Expression Real-Time PCR assays. Result was expressed as the threshold cycle (Ct). The relative quantification of the target transcripts was determined by the comparative Ct method (ΔΔCt) according to the manufacturer's protocol. The 2-ΔΔCt method was used to analyze the relative changes in gene expression. Control experiments were conducted without reverse transcription to confirm that the total RNA was not contaminated with genomic DNA. β-actin was used as an internal control gene in order to normalize.Total RNA was extracted using the TRIzol RNA Kit  according to the manufacturer's protocol. One μg of total RNA was used to generate cDNA using SuperScript II reverse transcriptase (Invitrogen). PCR was performed in triplicate using SsoFasrWestern blotting was performed as previously described , 37, witApoptosis was analyzed by nuclear staining with Hoechst 33258 (Invitrogen) . AnnexinCells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Knockdown experiments were performed 24 hours prior to idelalisib treatment using 300 pmole of siRNA. The control scrambled siRNA and siRNA for human p65 (sc-29410), and GSK3β (sc-35527) were from Santa Cruz Biotechnology. For stable transfection a shRNA-expressing plasmid that containing the p53-targeting sequence (CACCATCCACTACAACTACAT) , PUMA- tHCT116 cells were pre-treated with BAY11-7082 or GSK-3β knockdown, and then subjected to idelalisib treatment for another 6 hours. Nuclear fractionation was used to analyze NF-κB nuclear translocation. Nuclear extracts were isolated from cells using the NE-PER nuclear/cytoplasmic extraction kit (Thermo Fisher) according to the manufacturer's instructions, and analyzed by p65 Western blotting.ChIP with p65 antibody  was performed using the Chromatin Immunoprecipitation Assay Kit (Millipore) according to the manufacturer's instructions. The precipitates were analyzed by PCR using primers 5'-GTCGGTCTGTGTACGCATCG-3' and 5'-CCCGCGTGACGCTACGGCCC-3' to amplify a PUMA promoter fragment containing putative κB sites .6 cells in 0.2 mL of medium were implanted subcutaneously on the back of athymic nude female mice. After tumor growth for 7 days, mice were treated with daily with idelalisib at 30 mg/kg by oral gavage for 10 consecutive days. Tumor growth was monitored by calipers, and tumor volumes were calculated according to the formula ½ × length × width2. Mice were euthanized when tumors reached ~1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin. TUNEL and active caspase 3 immunostaining was performed on 5 μM paraffin-embedded tumor sections, by using an AlexaFluor 488-conjugated secondary antibody (Invitrogen) for signal detection.All animal experiments were performed according to the related ethics regulations of Liaoning University of Traditional Chinese Medicine. HCT116 cells were harvested, and 4 × 10P values were calculated by the student's t-test and were considered significant if p < 0.05. The means ± one standard deviation (s.d.) is displayed in the figures.Statistical analyses were carried out using GraphPad Prism IV software.