These Ca2+ signals originate from the endoplasmic reticulum (ER) and regulate different cellular processes like metabolism, fertilization, migration, and cell fate. A prime target for Ca2+ signals are the mitochondria. ER–mitochondrial Ca2+ transfer is possible through the existence of mitochondria-associated ER membranes (MAMs), ER structures that are in the proximity of the mitochondria. This creates a micro-domain in which the Ca2+ concentrations are manifold higher than in the cytosol, allowing for rapid mitochondrial Ca2+ uptake. In the mitochondria, the Ca2+ signal is decoded differentially depending on its spatiotemporal characteristics. While Ca2+ oscillations stimulate metabolism and constitute pro-survival signaling, mitochondrial Ca2+ overload results in apoptosis. Many chemotherapeutics depend on efficient ER–mitochondrial Ca2+ signaling to exert their function. However, several oncogenes and tumor suppressors present in the MAMs can alter Ca2+ signaling in cancer cells, rendering chemotherapeutics ineffective. In this review, we will discuss recent studies that connect ER–mitochondrial Ca2+ transfer, tumor suppressors and oncogenes at the MAMs, and chemotherapy.Inter-organellar communication often takes the form of Ca Currently, mitochondrial function has been implicated in apoptosis, autophagy, cell proliferation, cellular senescence, and migration6. Furthermore, mitochondrial function is impacted by the “state” of the mitochondrial network, which can range from highly connected to fragmented7. Nevertheless, mitochondria do not act as sole orchestrators of cellular processes. In fact, the mitochondrial network rather functions as a highly versatile signaling platform, closely connected to other cell organelles, like the endoplasmic reticulum (ER)8 and peroxisomes9. To allow for inter-organellar cross-talk, the different organelles are often located in close proximity to each other11, like the ER and the mitochondria, which are connected through mitochondria-associated ER membranes (MAMs). These MAMs are defined as ER membranes that are in close apposition (10–50 nm) to the mitochondria and were first isolated as a distinct entity in the early 1990s13. In recent years, MAMs were shown to contribute to various cellular functions like metabolism, autophagy, lipid synthesis but also cell survival and cell death18. In this sense, the MAMs, like mitochondria, are highly dynamic signaling hubs where signals from different cellular pathways converge and are integrated21.Mitochondria do not only fulfill the function of powerhouse of the cell but their function also encompasses more than merely providing the cell with ATP2+23. While [Ca2+] in the cytosol is maintained at low levels under resting conditions, the bulk of intracellular Ca2+ is confined in the ER22. Ca2+ is predominantly released from the ER via the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), which is gated by IP324, or the ryanodine receptor (RyR)25. However, Ca2+ accumulation into the mitochondrial matrix requires Ca2+ transport across the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). At the OMM, Ca2+ transport is mediated via the high-conductance voltage-dependent anion channel 1 (VDAC1), while at the IMM, Ca2+ transport is mediated via the mitochondrial Ca2+ uniporter (MCU), the pore-forming unit in the MCU complex, consisting of MCU itself and its regulators28. For a detailed description of MCU regulation we would like to refer to refs. 30. The MAMs play an important role in mitochondrial Ca2+ uptake, since they provide a Ca2+ micro-domain, where Ca2+ levels are higher than in the bulk cytosol15. This is necessary to sustain ER–mitochondrial Ca2+ signaling since the MCU has a low affinity for Ca2+. Thus, the MAMs allow for efficient, ‘quasi-synaptic’ mitochondrial Ca2+ uptake upon ER Ca2+ release through the formation of a micro-domain31. This emphasizes the importance of the MAMs as a signaling hotspot.One of the signals transferred between ER and mitochondria at the MAMs is the ubiquitous second messenger Ca2+ signals are decoded differentially depending on their spatiotemporal characteristics  cycle, including pyruvate, isocitrate, and α-ketoglutarate dehydrogenases32, while MCU transcription is controlled by the cAMP-responsive element binding protein, a Ca2+-dependent transcription factor33. Cells can also fine tune the level of Ca2+ oscillations that drive mitochondrial bioenergetics via a redox-nano-domain34. Mitochondrial Ca2+ uptake triggers K+- and H2O-influx into the mitochondrial matrix, leading to cristae compression and H2O2 release at the MAMs. This provides positive feedback on IP3Rs, enhancing their ability to sustain Ca2+ oscillations34. Other contributions of Ca2+ to cell metabolism are the stimulation of complex III of the electron transport chain, as well as stimulation of the ATP synthase and the adenine nucleotide translocase35.Mitochondrial Casee Fig. . For exa2+ oscillations in the cytosol can also modify mitochondrial metabolism indirectly by activating ARALAR, a mitochondrial glutamate/aspartate transporter playing a central role in the malate/aspartate shuttle, which is strongly dependent on cytosolic Ca2+ signaling36. Ca2+ binds to ARALAR and activates ARALAR-mediated glutamate and NAD(P)H transport into the mitochondria38. Also, pyruvate production is Ca2+ dependent and is linked to the malate/aspartate shuttle, providing pyruvate to the mitochondria40. In addition, there is tissue-specific regulation, which determines the threshold for mitochondrial Ca2+ influx and its effects41. Liver cells and cardiomyocytes contain a high and a low ratio of mitochondrial Ca2+ uptake 1 (MICU1)/MCU, respectively, with MICU1 (together with MICU2) being an MCU regulator which affects the cooperativity of mitochondrial Ca2+ uptake43. Therefore liver cells display a high cooperativity of mitochondrial Ca2+ uptake, while cardiac cells display a low cooperativity. This prevents the occurrence of mitochondrial Ca2+ signals in response to short-lasting Ca2+ transients and thus avoids mitochondrial Ca2+ overload even when the heart beats at high frequency41. Recently, it was shown that apart from affecting the cooperativity of mitochondrial Ca2+ uptake, MICU1, together with its paralog MICU2, inhibits the MCU at cytosolic Ca2+ concentrations lower than ~600 nm, thereby determining the relatively high threshold for MCU-mediated Ca2+ uptake44. Compared to wild-type cells, loss of MICU1, which also results in loss of MICU2, lowers the threshold for MCU-mediated mitochondrial Ca2+ uptake to about 200 nM Ca2+. Compared to mitochondria lacking both MICU1 and MICU2, mitochondria containing MICU1 but not MICU2 display a higher threshold for mitochondrial Ca2+ uptake (~350 nM Ca2+), indicating that MICU1 by itself can inhibit MCU. These observations indicate that MICU1 does not only control MCU cooperativity43, but can also function as a gatekeeper of MCU44. Moreover, MICU2 requires MICU1 to regulate MCU. MICU1’s function as a gatekeeper is also important in vivo to prevent mitochondrial Ca2+ overload, as evidenced in MICU1-knockout animals, developing ataxia and muscle fatigue associated with elevated mitochondrial Ca2+ levels and reduced ATP levels45. Finally, MICU1 and MICU2 were shown to bind the mitochondrial lipid cardiolipin, facilitating membrane anchoring of the complex and the fine-tuned Ca2+-dependent regulation of the MCU by MICU1 and associated factors, like EMRE44.Ca2+ release from the ER causes mitochondrial Ca2+ overload, resulting in opening of the mitochondrial permeability transition pore (mPTP)47, mitochondrial swelling, and subsequent cell death  that triggers the opening of the mPTP52. In addition to this, recent evidence emerged that mitochondrial Ca2+ could directly target the mPTP, resulting in a conformational change leading to its opening and to subsequent mitochondrial swelling53.On the other hand, massive Caath Fig. 48–51. Se2+ signaling at the MAMs, e.g. the IP3R and VDACs55, whereas others alter ER–mitochondrial Ca2+ signaling by acting through modulation of these Ca2+-transport systems, e.g. promyelocytic leukemia protein (PML)56 or phosphatase and tensin homolog (PTEN)57. Other proteins modify the characteristics of the MAMs (e.g. their distance to the mitochondria), like the ER–mitochondria tethers mitofusin-2 (Mfn-2)59, the protein kinase RNA-like endoplasmic reticulum kinase (PERK)60, and the spacer protein fetal and adult testis expressed 1 (FATE1)61. We will focus on the proteins involved in ER–mitochondrial Ca2+ transfer at the MAMs, yet for a more extensive list of proteins present at the MAMs, we refer to ref. 13. For further insights on the role of Ca2+-transport systems in cell death and survival, we refer to ref. Given its critical role in cell fate and survival, the composition and functional properties of the MAMs have to be carefully regulated and controlled. This occurs through a distinct set of proteins with a variety of cell biological properties and functions. Some of these proteins are directly involved in Ca2+ transfer occurs at the MAMs, it is not surprising that several intracellular Ca2+-transport systems reside at the MAMs. Both the IP3R and VDAC1 can be found at the MAMs, where they are connected to each other via glucose-related protein of 75 kDa (GRP75)55. Interestingly, isoform-specific functions for these channels at the MAMs have been discovered. Overexpression of VDAC1, VDAC2, as well as VDAC3 increased mitochondrial Ca2+ uptake, yet pro-apoptotic Ca2+ signals were only enhanced by VDAC1 overexpression63. Moreover, VDAC1 is the only VDAC isoform that co-immunoprecipitated with the IP3R63. Similarly, IP3R3 seems to be the isoform that preferentially transmits pro-apoptotic Ca2+ signals to the mitochondria via the MAMs65. Not only Ca2+-release channels are located at the MAMs, the sarco-/endoplasmic reticulum Ca2+ ATPase (SERCA) pump localizes to the MAMs as well68. SERCA pumps can influence the properties of the MAMs since their activity determines how fast Ca2+ is cleared from the micro-domain69. Moreover, SERCA pumps critically affect apoptotic sensitivity by controlling the steady-state filling level of the ER Ca2+ stores, which is determined by the balance between ER Ca2+ leak and ER Ca2+ uptake72. Also for SERCA, isoform-specific functions exist: ER stress causes induction of SERCA1T, a truncated splice-variant of SERCA1. SERCA1T is a determinant of ER leakiness, thereby promoting Ca2+ transfer to the mitochondria and thus supporting pro-apoptotic Ca2+ signaling74.Since ER–mitochondrial Ca2+-signaling proteins critically determine the cell’s Ca2+-signaling properties. Furthermore, ER–mitochondrial Ca2+ signaling is fine-tuned by various oncogenes and tumor suppressors76. These proteins may induce post-translational modifications that alter the Ca2+-signaling properties of proteins at the MAMs77. Particularly, phosphorylation of the IP3R is a critical factor: Akt/protein kinase B (PKB) suppresses IP3R-mediated Ca2+ release through phosphorylation78, while tumor suppressors PTEN (direct dephosphorylation of IP3R)57 and PML (indirect dephosphorylation via sequestration of protein phosphatase 2A (PP2A) and subsequent Akt/PKB inhibition)56 counteract this. Also, SERCA is a target for post-translational modification: p53 changes SERCA’s oxidative state, promoting its ER Ca2+-uptake activity and thus altering the net flux of Ca2+ released from the ER80.As will be discussed further on, expression levels of the Ca2+-transport proteins like IP3Rs and VDAC1 at the MAMs and tumor suppressors or oncogenes influences ER–mitochondrial Ca2+ transfer. Notably, several Bcl-2-protein family members, critical regulators of apoptosis, were shown to be present at the MAMs82 and can modify IP3R-mediated Ca2+-release84. Bcl-2, an anti-apoptotic protein of the Bcl-2-protein family, inhibits the IP3R directly by binding with its Bcl-2 homology (BH) 4 domain to the central, modulatory domain of the IP3R87. The BH4 domain also participates in overall stability of the Bcl-2 protein, affecting its IP3R-inhibitory properties88. Bcl-2’s transmembrane domain is necessary for efficient in cellulo IP3R inhibition89. Indirectly, Bcl-2 changes IP3R activity by providing a docking place at the IP3R for protein phosphatase 1 (PP1), which inhibits IP3R function by dephosphorylating the receptor90. Bcl-2 also regulates IP3R function by docking dopamine- and cAMP-regulated phosphoprotein of 32 kDa, a PP1 inhibitor, and the protein phosphatase calcineurin in a complex on the IP3R91. Thus, IP3R activity is regulated by a negative feedback mechanism that prevents excessive, pro-apoptotic Ca2+ release from the ER. Besides Bcl-2, also Bcl-Xl and Mcl-1 can modulate IP3R activity94. An extended discussion on the modulation of Ca2+ signaling by Bcl-2-protein family members can be found elsewhere95.In addition, complex formation between Ca3Rs, Bcl-2-family members can also target mitochondrial Ca2+-transport systems, including VDAC1. VDAC1/Bcl-2 complex formation inhibits VDAC1’s function in mitochondrial Ca2+ transport97. The BH4 domain of Bcl-2 appeared to play a critical role in VDAC1 regulation98. Follow-up work revealed that the BH4 domain of Bcl-Xl is more effective in targeting and modulating VDAC1 than the BH4 domain of Bcl-281. Consequently, while both the BH4 domains of Bcl-2 and Bcl-Xl could prevent mitochondrial Ca2+ uptake, BH4-Bcl-2 acted at the level of IP3Rs, while BH4-Bcl-Xl acted at the level of VDAC181. To summarize, ER–mitochondrial Ca2+ transfer at the MAMs can be influenced through IP3R-mediated Ca2+ release, VDAC1-mediated mitochondrial Ca2+ uptake, or modulation of the SERCA activity.Besides the IP2+ transfer is altered by the number of MAMs and the distance between the ER and the OMM at the MAMs100. Proteins that hold both membranes together are typically called tethers. An example of a tether at the MAMs is the GTPase Mfn-2 involved in mitochondrial fusion. Mfn-2 tethers ER and mitochondria through homo- and heterotypic interactions with Mfn-2 and Mfn-1, respectively101. The importance of tethering was shown in Mfn-2-knockout cells, where ER–mitochondrial Ca2+ transfer was severely reduced101. Yet, the role of Mfn-2 as a tether has been debated, since another study showed that ablation of Mfn-2 does not impair the ER–mitochondrial connection but contrarily, tightens it101. Here, Mfn-2 was proposed as a spacer that increases the distance at the MAMs and reduces ER–mitochondrial signaling. A typical spacer is FATE1, a cancer testis antigen, which is normally only expressed in the testis61. However, in certain cancers, FATE1 becomes upregulated and causes MAMs alterations61.In addition to this, ER–mitochondrial Ca2+ signaling impart an enormous flexibility to the cell concerning fine-tuning cellular processes in response to internal and external stimuli. However, this sensitive system can be hijacked to drive malignant transformations in the cell6. It is known that several types of cancer cells undergo an extensive rewiring of their Ca2+-signaling machinery, favoring oncogenesis103. At the level of the ER and the mitochondria, expression levels of Ca2+-signaling proteins, including VDAC1, IP3R, and SERCA, are often altered in cancer cells. For instance, VDAC1 expression levels are correlated with tumor growth and invasion in several types of cancer, e.g. non-small cell lung cancer and cervical cancer105. In this regard, recently, genetic disruption of VDAC1 in cells from cancer xenograft models displayed decreased mitochondrial membrane potential and ATP content with a consequent low migration rate and tumor regression107. Another example includes IP3R1 downregulation in bladder cancer cells, which attenuates cisplatin-mediated apoptosis through a decrease in ER–mitochondrial Ca2+ uptake, preventing mitochondrial Ca2+ overload109. Remodeling of Ca2+ signaling in tumorigenesis is also documented by the significant reduction or loss of the SERCA3 isoform in transformed colonic epithelial cells110. Different mechanisms may be responsible for the change in expression levels. Recently, two novel mechanisms dysregulated in several cancer types have been discovered to impact the proteasomal turnover and thus stead-state expression levels of IP3R3 and the apoptotic sensitivity of cells111. (i) The tumor suppressor PTEN competes with F-box/LRR-repeat protein 2 (FBXL-2), an E3 ubiquitin ligase component belonging to the Skip-Cullin1-F-box protein family112, for binding to IP3R3, thereby slowing down FBXL-2-mediated proteasomal degradation of IP3R3113. This represents a novel mechanism by which loss of PTEN allows cancer cells to evade apoptosis, since pro-apoptotic mitochondrial Ca2+ transfer becomes impaired due to downregulation of the IP3R3. (ii) The tumor suppressor BRCA1-associated protein 1 (BAP1), a deubiquitylating enzyme, promotes ER–mitochondrial Ca2+ transfer by stabilizing the IP3R3114. BAP1 function is particularly impaired during prolonged environmental stress, associated with acquired inactivating mutations in BAP1 genes. Loss of BAP1 results in IP3R3 downregulation, hampering the effective apoptotic clearance of damaged cells and favoring oncogenesis and malignant cell survival.The different possible layers of regulation of Ca115. As such, miR-25, targeting MCU, was overexpressed in colon cancer cell lines and tumor samples, decreasing MCU expression compared to non-tumorigenic cells. Moreover, miR-25 overexpression in HeLa cells reduced mitochondrial Ca2+ accumulation, resulting in apoptosis resistance. In contrast, antagonizing miR-25 expression using antagomirs re-sensitized colon cancer cells to Ca2+-dependent apoptotic stimuli, like H2O2 and ceramide. Interestingly, MCU may prevent tumor cell survival in an early stage, but can become a pro-malignant factor in late-stage tumors, like triple negative breast cancer cells116. These cells express high levels of MCU, correlating with tumor size and lymph node infiltration, which negatively impact survival outcome. The mechanisms involved MCU-dependent uptake of Ca2+ into the mitochondrial matrix and subsequent generation of ROS that stabilized hypoxia-inducible factor-1α, a transcription factor driving the expression of genes involved in cancer migration and invasion117.While MCU is not residing at the MAMs, its expression can be controlled in a tumor-specific manner, e.g. via microRNAs (miR)2+-signaling modulation108. A striking example of this is apoptotic resistance. Since mitochondrial Ca2+ overload is involved in apoptotic cell death, modifying ER–mitochondrial Ca2+ transfer at the MAMs alters apoptotic sensitivity48. Cancer cells can gain resistance against cell death, e.g. by overexpressing proteins that suppress IP3R-mediated Ca2+ signaling, like Akt78, or by increasing the intermembrane distance at the MAMs (e.g. FATE1), thereby rendering ER–mitochondrial Ca2+ transfer less efficient61. These mechanisms are not only supporting basic cancer cell characteristics, but also underlie resistance against chemotherapy. Cell death induction strategies still play a central role in the fight against cancer. While selective oncogene inhibitors, like venetoclax/ABT-199 for Bcl-2 inhibition, emerged and entered the clinic118, chemotherapy remains a very effective way to eradicate tumor cells by triggering cell death119.Furthermore, oncogenes like Akt/PKB and FATE1, and tumor suppressors like PML and PTEN, can play additional roles in the development of cancer via Ca2+ signaling, Ca2+ fluxes across the plasma membrane can affect cancer properties as well. We would like to refer to other excellent reviews regarding this topic122, as in this section, we would like to focus on chemotherapeutic drugs that act on Ca2+ signaling at the MAMs.Besides ER–mitochondrial Ca2+ signaling appears to be a major contributor to the cytotoxic effects of chemotherapy. Many chemotherapeutic agents trigger a rapid onset of cytosolic [Ca2+] rises123. Furthermore, shifts in cytosolic [Ca2+] have been proposed as early markers for cytotoxicity in cells in response not only to H2O2 or staurosporine but also to chemotherapeutics like gossypol or arsenic trioxide (ATO)123. The mechanism of these early cytosolic [Ca2+] elevations is not always fully understood, but may in part depend on the presence of p53. Upon chemotherapeutic treatment, extra-nuclear p53 can accumulate at the ER membranes where it binds SERCA and activates its ER Ca2+-uptake activity125. Thus, SERCA activation will augment the ER Ca2+-store content, overfilling the ER with Ca2+ and thus increasing the likelihood for spontaneous ER Ca2+-release events126. However, the occurrence of shifts in cytosolic [Ca2+] may not be a general phenomenon, as on-target Bcl-2 inhibitors, like ABT-737127 and venetoclax/ABT-199128, do not trigger these early cytosolic [Ca2+] elevations, even not in cancer cells that are dependent on Bcl-2 for their survival. The reason for these varying responses are not fully understood, but may actually relate to the mechanism of action of the drug applied and in particular whether p53 is involved.Ca2+ signaling. These chemotherapeutics are summarized in Table Here, we will discuss the chemotherapeutic agents that act via ER–mitochondrial Ca130. APL patients are mostly treated by a combination of all-trans retinoic acid and ATO therapy, which stimulates APL cell differentiation by triggering proteasomal degradation of the PML/RARα fusion protein133. This treatment approach results in high APL cure rates, reflected by high complete remission and overall survival percentages132.Acute promyelocytic leukemia (APL) is almost always characterized by a t chromosomal translocation, resulting in a PML/retinoic acid receptor (RAR) α fusion protein that hinders the differentiation of hematopoietic cells by inhibiting gene transcription2+ signaling, thereby repressing autophagy in cancer cells17. Autophagy is an important pro-survival pathway in malignant cells that experience oncogenic stress, as missing nutrients can be delivered to the cells via this process134. The tumor suppressor PML, which is localized at the MAMs, represses autophagy by promoting ER–mitochondrial Ca2+ transfer and mitochondrial respiration17. Hence, PML is often downregulated in cancer cells135. Loss of PML results in reduced IP3R-mediated Ca2+ transfer from the ER to the mitochondria, leading to decreased mitochondrial Ca2+ levels, thereby diminishing mitochondrial respiration and ATP production17. This triggers activation of AMP-activated protein kinase, which stimulates pro-survival autophagy by a mechanism involving mechanistic target of rapamycin and unc-51-like kinase 1 pathways137. Interestingly, this autophagic process is repressed in APL cells treated with ATO17. Short-term treatment of these cells with ATO promotes the selective degradation of the PML/RARα fusion protein, but not of PML. Furthermore, exposure to ATO increases the PML levels at ER–mitochondria contact sites in a p53-dependent manner  and ABT-199 (venetoclax), which solely targets the hydrophobic cleft of Bcl-2145. While these drugs are specifically developed to suppress the canonical, anti-apoptotic function of the Bcl-2-protein family members at the mitochondria, it seems that their intracellular effects are more complex.BH3 mimetics are a class of anti-cancer drugs inhibiting the function of anti-apoptotic Bcl-2-protein family members like Bcl-2, Bcl-Xl, and Mcl-1151. Dysregulation of intracellular Ca2+ homeostasis might underlie ABT-737- and ABT-263-induced thrombocytopenia, as addition of ABT-263 to platelets triggered an acute rise in cytosolic Ca2+ levels149. However, a direct link between deranged Ca2+ signaling and platelet dysfunction was not provided since this effect was only observed upon addition of relatively high ABT-263 concentrations (10 µM), whereas platelet function was already decreased at much lower concentrations (100 nM–1 µM)149. Furthermore, prolonged treatment of platelets with ABT-263 and ABT-737 depleted the intracellular Ca2+-storage organelles152. However, it was not clear whether the effects on Ca2+ signaling were the cause of platelet apoptosis or whether they were the consequence of platelets being in late-stage apoptosis due to Bcl-Xl inhibition. Our lab excluded a direct impact of ABT-737 on intracellular Ca2+ signaling, since ABT-737 application did not affect thrombin-induced Ca2+ signaling in platelets nor ATP-induced Ca2+ signaling in HeLa cells127. Moreover, SERCA activity and IP3R-mediated Ca2+ release were unaffected by ABT-737. These results argue against a proximal role of Ca2+-signaling dysregulation in platelet dysfunction and apoptosis induced by ABT-737.Thrombocytopenia is an important side effect caused by ABT-737 and ABT-263, since these BH3 mimetic drugs inhibit the function of Bcl-Xl, which is essential for platelet formation and survival2+ homeostasis153. In several human and mouse cell models, ABT-199 did not trigger cytosolic Ca2+ release events by itself nor did it affect agonist-induced IP3R-mediated Ca2+ signaling128. Also, clearance of Ca2+ from the cytosol after agonist application was not affected. Furthermore, ABT-199 did not interfere with the inhibition of IP3R caused by overexpression of Bcl-2128. Nevertheless, it seems that there is an interplay between ABT-199-induced cell death in Bcl-2-dependent cancer cells and basal Ca2+ signaling, since chelating intracellular Ca2+ using BAPTA-AM enhanced ABT-199-induced cell death128. The mechanisms underlying this phenomenon remain unclear, but might be due to downregulation of anti-apoptotic Bcl-2-family members or upregulation of pro-apoptotic Bcl-2-family members. Furthermore, Bcl-2-dependent cancer cells can be sensitized towards ABT-199 by the application of BIRD-2154, a BH4-domain inhibitor of Bcl-2 that triggers toxic Ca2+-release events and apoptosis in various cancer cells, including chronic lymphatic leukemia, diffuse large B-cell lymphoma, multiple myeloma, follicular lymphoma, and lung cancer cells156. Further, it appears that BIRD-2 upregulates Bim in a Ca2+-dependent manner, thereby likely accounting for an increased sensitivity of the cells towards BH3 mimetics like ABT-199 application157. As such, BIRD-2 and ABT-199 can act synergistically to trigger cell death in Bcl-2-dependent cancers. Finally, it appears that cancer cells that are less sensitive to BH3 mimetics are more sensitive to BIRD-2 and vice versa157.On the other hand, for ABT-199/venetoclax, which only targets the hydrophobic cleft of Bcl-2, no evidence was found for a perturbation of Ca158. In the latter, ABT-737 treatment increased the cisplatin-induced growth inhibition and apoptosis in cisplatin-resistant cells, hence restoring their sensitivity to the chemotherapeutic agent143. Combination therapy with cisplatin and ABT-737 of the cisplatin-resistant ovarian cancer cells increased the number of ER–mitochondria contact sites induced by cisplatin, stimulating Ca2+ transfer from the ER to the cytosol and the mitochondria  as well as in ovarian cancer cells2+ homeostasis. HA14-1 and stabilized HA14-1s can deplete ER Ca2+ stores by inhibiting the SERCA Ca2+ pump159. In a separate study, both HA14-1 and BH3I-2′ trigger Ca2+ release from the ER of pancreatic acinar cells through IP3R- and RyR-mediated mechanisms, elevating cytosolic Ca2+ levels, a feature that contributes to their cell death properties160. Here, it was proposed that dissociation of Bax from Bcl-2 using these drugs sensitize IP3Rs and RyRs to cytosolic Ca2+. Excitingly, the ability of HA14-1 and BH3I-2′ to increase cytosolic Ca2+ levels in pancreatic acinar cells was strictly dependent on the presence of Bax, while the presence of Bcl-2 and Bak was not critical for this process. It was proposed that Bax, released from Bcl-2, can induce Ca2+ leak from the ER, either by itself or by acting on ER Ca2+-leak channels like IP3Rs161. Yet, further work using selective Bcl-2 inhibitors is needed to validate this model. A detailed discussion on the role of BH3 mimetics and Ca2+ signaling is provided elsewhere162.Of note, earlier versions of Bcl-2-inhibiting molecules can dysregulate intracellular Ca165. It is known as a multi-target agent exhibiting antioxidant, anti-inflammatory, and immunomodulatory activities. This pleiotropic compound affects cell proliferation, differentiation, apoptosis, and autophagy and attenuates many age-related chronic complications, such as metabolic, cardiovascular, and neurodegenerative diseases165. Furthermore, resveratrol has been used as a chemopreventive and chemotherapeutic agent in many types of cancer165. For instance, it has been used in clinical trials conducted in patients with colon, colorectal, and gastrointestinal cancers as well as in a trial examining the effects of resveratrol in the prevention of cancer in healthy participants163. Remarkably, resveratrol functions as a specific anticancer agent with limited toxicity in normal cells163. However, the exact mechanism by which resveratrol specifically kills cancer cells remains unclear.Resveratrol is a natural polyphenol produced in response to stressful conditions by various plant species. It is found in several foodstuffs, including grapes, mulberries, and peanuts, and has been attributed beneficial health effects since the early 1990s166. Cancer cells are characterized by a very high ATP demand at the ER because of immense protein folding activities going on at this organelle168. Therefore, tethering of the ER and the mitochondria, which produce ATP via the ATP synthase localized in the IMM, is strongly enriched in cancer cells, warranting high ATP levels in the proximity of the ER170. However, resveratrol, which acts as an inhibitor of the F1 subunit of the ATP synthase174 exploits the enhanced ER–mitochondria coupling in malign cells to kill these cells exclusively , an enzyme that protects cells against the harmful effects of free cholesterol by transforming it into cholesterol esters. Because of SOAT1 inhibition, mitotane therapy leads to the accumulation of toxic lipids, including free cholesterol and oxysterols, inside ACC cells, which triggers lipid-induced ER stress190. Moreover, in ACC tissue samples, SOAT1 expression correlated with the response to mitotane treatment.Another chemotherapeutic drug that acts on lipid metabolism is mitotane, a derivative of the insecticide dichlorodiphenyl-trichloroethane. Mitotane is the only chemotherapeutic drug approved for the treatment of adrenocortical carcinoma (ACC), one of the deadliest endocrine malignancies61. Under physiological conditions, FATE1 expression is restricted to the testis and adrenal gland, while FATE1 overexpression is observed in a variety of cancers192. In ACC cells, FATE1 expression is controlled by the steroidogenic factor-1 (SF-1), a transcription factor that is important for adrenal development and plays a role in the formation of adrenocortical tumors191. FATE1, localized at the MAMs where it uncouples the ER and mitochondria, decreases mitochondrial Ca2+ uptake in ACC cells61. In this way, FATE1 protects cancer cells from Ca2+-dependent apoptotic stimuli. Furthermore, FATE1 expression conferred mitotane resistance to ACC cells, when this chemotherapeutic drug was used in a dose that falls inside the therapeutic window for ACC patients, whereas knockdown of FATE1 in these cells increased the sensitivity to the drug61. FATE1 probably protects against mitotane-induced apoptosis because of its localization in the MAMs61. Mitotane inhibits the SOAT1 enzyme190, which is also localized in the MAMs, resulting in the accumulation of toxic cholesterol lipids. Hence, in the presence of FATE1 mitotane-mediated SOAT1 inhibition may be less efficient (Fig. 61. Interestingly, FATE1 expression in ACC tumor cells can even be used as a prognosis indicator since FATE1 expression is inversely correlated with the overall survival of ACC patients61.Interestingly, mitotane-induced apoptosis in ACC cells also depends on FATE1 expressionent Fig. 61. Inter2+-encoded messages between the ER and the mitochondria. This ER–mitochondrial Ca2+ exchange can be altered during cancer development to promote cancer hallmarks like evasion of apoptosis, excessive cell proliferation, and a metabolic rewiring. In addition, many chemotherapeutics act via Ca2+ signaling at the MAMs (Fig. 2+ transfer. In this sense, chemotherapeutics that modify ER–mitochondrial Ca2+ signaling can be used to increase the response of cancer cells towards therapeutics that harbor a Ca2+ component in their working mechanism.In conclusion, MAMs form important intracellular signaling platforms, allowing for CaAMs Fig. . Moreove


Weakening this physical linkage may reduce Ca2+ uptake into mitochondria, while fortifying these organelle contact sites may promote mitochondrial Ca2+ overload and cell death. Small conductance Ca2+-activated K+ (SK) channels regulate mitochondrial respiration, and their activation attenuates mitochondrial damage in paradigms of oxidative stress. In the present study, we enhanced ER–mitochondrial coupling and investigated the impact of SK channels on survival of neuronal HT22 cells in conditions of oxidative stress. Using genetically encoded linkers, we show that mitochondrial respiration and the vulnerability of neuronal cells to oxidative stress was inversely linked to the strength of ER–mitochondrial contact points and the increase in mitochondrial Ca2+ uptake. Pharmacological activation of SK channels provided protection against glutamate-induced cell death and also in conditions of increased ER–mitochondrial coupling. Together, this study revealed that SK channel activation provided persistent neuroprotection in the paradigm of glutamate-induced oxytosis even in conditions where an increase in ER–mitochondrial coupling potentiated mitochondrial Ca2+ influx and impaired mitochondrial bioenergetics.Alterations in the strength and interface area of contact sites between the endoplasmic reticulum (ER) and mitochondria contribute to calcium (Ca Disrupted Ca2+ homeostasis can lead to mitochondrial Ca2+ ([Ca2+]m) overload, and subsequently to an impairment of mitochondrial energy metabolism and respiration7. Under physiological conditions however, [Ca2+]m is a prerequisite for aerobic energy metabolism through the regulation of oxidative phosphorylation (OXPHOS) and mitochondrial ATP production9.Multiple lines of evidence indicate that the etiologies of neurodegenerative disorders, such as Alzheimer’s disease (AD) or Parkinson’s disease (PD) are strongly associated with common features of neuronal damage such as dysregulation of calcium , thereby facilitating Ca2+ transfer between ER and mitochondria through mitochondrial voltage-dependent anion channels (VDAC) and ER-located inositol-1,4,5-trisphosphate receptors (IP3R), which are physically connected by glucose-regulated protein 75 (GRP75)12. Mutations in MAM-associated proteins have been identified to either enhance or reduce ER–mitochondrial coupling (EMC), thereby leading to dysregulation of MAM interfaces and progressive neuronal degeneration as shown in models of AD and amyotrophic lateral sclerosis15.Close spatial interactions between the endoplasmic reticulum (ER) and mitochondria are essential for rapid and sustained m uptake in the presence of the linkers and their pharmacological inducer rapamycin. In order to study fast changes in [Ca2+]m uptake induced by strengthening EMC, we utilized a system to perform real-time [Ca2+]m uptake measurements in HEK293T cells transfected with the linkers and a mitochondrial GFP-aequorin21. Up to 80% of HEK293T cells on average were transfected with the wild type (mtGAwt) or a Ca2+-binding deficient mutant aequorin version (mtGAmut) serving as an internal control  was unchanged, indicating the specific binding of Ca2+ to the mtGAwt construct , was recorded in real-time following the application of 200 nM rapamycin over a period of 2 h. In cells transfected with the Flipper control construct Fig.  but seems Figure .Fig. 2En2+]m influx, affected ATP production, and impaired the reserve respiratory capacity, without affecting basal respiration, cell viability, or proliferation.Taken together, we show that promoting EMC specifically amplified m load are critical factors to maintain mitochondrial function, with alterations in these processes promoting mitochondrial dysfunction. For instance, enhanced EMC provoked by the saturated fatty acid palmitate disrupted intracellular Ca2+ homeostasis, thereby promoting hepatotoxicity32, and enhanced VDAC-mediated [Ca2+]m influx amplified cell death induction in HeLa cells33. Here, we utilized mitochondria-targeted GFP-aequorin21 measurements to confirm that strengthening EMC by inducing heterodimerization of the linkers in HEK293T cells specifically increased Ca2+ uptake into the mitochondria. Ca2+ signaling through the MAM interface also determines the cellular bioenergetic state, as shown for Ca2+ release through IP3R35 and the interaction of mitofusin 1 and mitofusin 236. We observed that the increase in [Ca2+]m influx, elicited by heterodimerization of the EML, was accompanied by a decrease in ATP production and in the mitochondrial reserve respiratory capacity. In contrast, in a model of ER stress, EMC was increased at early stages following ER stress induction, which was accompanied by an initial increase in mitochondrial respiration before it was attenuated concomitantly with the onset of cell death (20 h)34. The reason for these different effects may be that EMC induced by the bifunctional linkers was a rather fast and highly efficient process occurring within minutes compared to the ER stress-induced increase in EMC (1–4 h) observed by Bravo and colleagues34. Thus, we propose that basal mitochondrial respiration was stable without an initial increase due to the speed and strength of the artificial linkage induced by the linkers.ER–mitochondrial Ca2+ homeostasis, and stress-induced Ca2+ dysregulation in both organelles is detrimental38. To this end, we investigated cell survival following stress induction in HT22 cells expressing the bifunctional linkers to establish a stable platform for increased ER–mitochondrial interactions. Glutamate-induced cell death, starting approximately 12 h following induction, was more pronounced in cells transfected with the linkers compared to control cells. Thus, our findings indicate that EMC is a critical factor that potentiates neuronal damage generated by glutamate exposure.The intensity of ER–mitochondrial associations therefore also regulates local Ca2+ signaling and mitochondrial function40. Despite their localization at the plasma membrane, recent studies have identified intracellularly expressed SK channels in the ER membrane and the IMM41 where they provide protection against ER stress and/or mitochondrial demise. Here, we show that activation of SK channels by the pharmacological SK2/3 channel activator CyPPA was able to protect against glutamate toxicity even in conditions of enhanced EMC that was associated with increased [Ca2+]m uptake and impaired mitochondrial respiration. Interestingly, the overexpression of either wild-type or mitoSK2 channels enhanced the neuroprotective effects of CyPPA following glutamate treatment. Wild-type SK2 channels partially co-localized with the ER16 suggesting a contribution of SK channels from both, ER and mitochondria, to signaling along the MAM interface which might contribute to the observed protection during EMC enhancement. This concept is further supported by the fact that the physical association via IP3R1–VDAC1 interactions was not changed by SK channel activation and/or overexpression. These results highlight that SK channel activation is a promising approach to prevent neurodegeneration in conditions of glutamate toxicity even if neuronal cells are facing additional stress, such as increased [Ca2+]m flux along the ER–mitochondrial axis and reduced mitochondrial respiration.SK channels, which are involved in controlling synaptic plasticity and neuronal firing, preserve cell survival in multiple disease paradigms by regulating Ca2+]m uptake and impaired mitochondrial bioenergetics, thereby potentiating neuronal cell death initiated by glutamate. SK channel activation by CyPPA, known to provide protection in different in vitro models of neurodegeneration, conferred protection against glutamate toxicity even when stress was pronounced due to an increase in EMC. These findings open a new platform to study SK channel-mediated protection in conditions of oxidative stress and mitochondrial dysfunction that are related to changes in ER–mitochondrial signaling.In conclusion, we show that strengthening EMC using genetically encoded linkers created a stable platform for ER–mitochondrial interactions, which enhanced [Ca2. Plasmid transfection was performed using the attractene transfection reagent according to the manufacturer’s fast-forward protocol . HT22 cells were transfected with 1.2 µg plasmid DNA and grown in a 6-well plate for 48 h followed by re-seeding into the appropriate plate format for subsequent experiments. HEK293T cells were transfected with 400 ng plasmid DNA and grown in a white-walled 96-well plate for 48 h. HT22 cells were challenged with glutamate .HT22 cells were cultured in Dulbecco’s modified Eagle Medium  supplemented with 10% heat-inactivated fetal calf serum , 100 U/mL penicillin, 100 µg/mL streptomycin, and 2mM L-glutamine  at 37 °C and 5% CO42 (ACEA Biosciences). Cell impedance was normalized to the time of treatment , which is defined as the starting point (t = 0 h) of the experiment.Cell viability was determined based on the metabolic activity using the MTT assay at a final concentration of 0.5 g/L by incubation for 1 h at 37 °C, followed by removal of the MTT and at least 1 h incubation at −20 °C. After dissolving the resulting formazan in DMSO, the absorbance of each well was determined with the Synergy H1 Multi-Mode reader  at 570 nM and at 630 nM. Alternatively, cell viability was monitored in real-time with cell impedance measurements, using the xCELLigence system5 cells/well) were grown on Ø25 mm coverslips . After image acquisition in untreated conditions, rapamycin (100 nM) was applied, and images were taken again after 5 min and 10 min at 60× magnification, pixel size 6.5 μm × 6.5 μm, 2560 × 2160 pixels, speed 400 fps at 512 × 512 pixel. The software adopted for acquisition and integrated deconvolution was softWoRx (on Linux CentOS 6.3 platform). Image overlays were done using the ImageJ software.To evaluate ER–mitochondrial contact formation upon transfection with TOM70-FKBP-mRFP and either ER-9xFRB-CFP  or ER-Flipper-GFP (depicted as FL) followed by the rapamycin-induced heterodimerization, widefield fluorescence microscopy (DeltaVision Elite) studies were performed. Transfected HT22 cells  for 48 h in a clear bottom, white-walled 96-well plate . Briefly, mtGA constructs were reconstituted with native coelenterazine  in medium for 2 h. Cells were then washed with PBS followed by addition of 100 µL internal buffer . Ca2+ uptake was initiated by addition of 50 mM CaCl2 or 500 µM carbachol in internal buffer supplemented with 2.5 mM MgEDTA following background measurement for 5 s. Final sensor saturation was performed by addition of lysis buffer . Luminescence was recorded using the FluoStar OPTIMA plate reader . Each condition was measured at least in triplicate, and Ca2+ uptake was calculated as the total luminescent counts per second (L) from the time of stimulation (t = 5 s) until the time of final cell lysis (t = 30 s). Values were normalized to the initial value. For mtGAwt measurements, these values were additionally normalized to the total luminescence (t = 0 s until t = 45 s). The sensor was calibrated with increasing CaCl2 concentrations  resulting in a dose-dependent increase in the luminescent signal as determined by cps and the normalized luminescence .HEK293T cells were transfected with wild type and mutant mitochondrial GFP-aequorin . Prior to the measurement, the medium was removed and replaced by 180 μL assay medium containing 4.5 g/L glucose, 2 mM L-glutamine, 1 mM pyruvate (pH 7.35) for 1 h at 37 °C. The OCR was analyzed using the Seahorse XF Biosystem. Three baseline measurements  were recorded followed by injection of rapamycin (50–200 nM) or medium and measurement for 2 h (3/5/3). Following EMC, mitochondrial function was assessed by injection of 4 µM oligomycin (3/0/3), 50 µM DNP (3/0/3), and 150 nM rotenone, 1 µM antimycin A, and 50 mM 2-DG (3/0/3). After injection of each compound, the OCR was determined and values were normalized before injection into port A.43 was used to calculate N0 values which were normalized to N0 of Gapdh as an internal control. Data are acquired in duplicates from three independent experiments.CHOP and GAPDH expression were analyzed using quantitative real-time PCR (qPCR). Therefore, total RNA was extracted from HT22 cells transfected with TOM70-mRFP and either ER-Flipper GFP (FL) or ER-CFP (EML) and treated with 100 nM and 200 nM rapamycin for 6 h, using Trizol RNA extraction . The cDNA was synthesized from 500–1000 ng RNA using the Reverse Transcriptase System , and the following protocol: 10 min 25 °C, 45 min 42 °C, 5 min 99 °C. The qPCR was performed with SYBR Green  and the following protocol including a final step to generate the melting curve: 2 min 95 °C, 10 min 95 °C, 45× , 30 s 95 °C, 30 s 55 °C, 30 s 95 °C. The qPCR was performed in an Eco Illumina . For analysis, the LinRegPCR softwareChop (5′ CATACACCACCACACCTGAAAG 3′ and 3′ CCGTTTCCTAGTTCTTCCTTGC 5′) and Gapdh (5′ GGAGAGTGTTTCCTCGTCCC 3′ and 3′ ATGAAGGGGTCGTTGATGGC 5′).The following murine primers were used: 3R/VDAC1 complex at the MAM interface was performed, as previously described45. Briefly, following CyPPA treatment or transfection with GFP/SK2 constructs, HT22 cells cultured on 35 mm glass bottom dishes  were fixed with 4% PFA for 10 min, permeabilized using 0.3% PBS/TritonX-100 for 30 min, and saturated. The in situ PLA was performed according to the manufacturer’s protocol: VDAC1 (mouse anti-VDAC1 primary antibody) and IP3R1 (rabbit anti-IP3R1 primary antibody) were probed followed by addition of the secondary antibodies anti-mouse and anti-rabbit IgG (PLA probe MINUS and PLUS) conjugated to complementary oligonucleotide extensions. If target proteins were within 40 nM proximity, the oligonucleotides could hybridize with the connector oligonucleotides to form a circular DNA template, which was ligated and amplified to create a single-stranded DNA product. The DNA product is covalently attached to one of the proximity probes to allow detection of hybridized Texas red-labeled oligonucleotide probes. Each red fluorescent dot represents a site of IP3R1–VDAC1 interaction. Duolink II mounting medium containing DAPI 18  was used for mounting, and preparations were analyzed with a Zeiss inversed fluorescent microscope at 63× magnification. Quantification of signals (number of red dots per cell) was done using the BlobFinder software. Experiments were performed in triplicate, n = 25–30 pictures per conditions.An optimized in situ proximity ligation assay (PLA) targeting the IPt-test or Analysis of variance (ANOVA) and Scheffé’s test for multiple comparisons, unless otherwise stated. p-values indicating statistically significant differences between the mean values are defined as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.Statistical significance was assessed using the unpaired Student’s Supplementary figure LegendsSupplementary figure 1Supplementary figure 2Supplementary figure 3


RAS proto-oncogenes and their effectors are common in acute myeloid leukemia (AML); however, efforts to therapeutically target Ras or its effectors have been unsuccessful, and have been hampered by an incomplete understanding of which effectors are required for AML proliferation and survival. We investigated the role of Ras effector pathways in AML using murine and human AML models. Whereas genetic disruption of NRAS(V12) expression in an NRAS(V12) and Mll-AF9-driven murine AML induced apoptosis of leukemic cells, inhibition of phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) signaling did not reproduce this effect. Conversely, genetic disruption of RALB signaling induced AML cell death and phenocopied the effects of suppressing oncogenic Ras directly – uncovering a novel role for RALB signaling in AML survival. Knockdown of RALB led to decreased phosphorylation of TBK1 and reduced BCL2 expression, providing mechanistic insight into RALB survival signaling in AML. Notably, we found that patient-derived AML blasts have higher levels of RALB-TBK1 signaling compared to normal blood leukocytes, supporting a pathophysiologic role for RALB signaling for AML patients. Overall, our work provides new insight into the specific roles of Ras effector pathways in AML and has identified RALB signaling as a key survival pathway.Mutations that activate  NRAS and KRAS proto-oncogenes and genes that regulate Ras activation (i.e. PTPN11 and NF1) are frequently mutated in AML. Moreover, many common mutations in AML occur upstream of Ras (i.e. FLT3 ITD and KIT), and likely rely on Ras signaling for their oncogenic effects [The i.e. PTPN1 and NF1 effects , 5. DespRAS mutations are among the most common molecular alterations in human cancer, and thus has been the focus of intense interest for drug development [RAS genes, most commonly involving amino acid substitutions at codons 12, 13, and 61 impair GTP hydrolysis, thereby leading to constitutive activation of Ras effector pathways and cellular transformation [Oncogenic elopment . Ras proelopment . Ras actelopment . Oncogenormation . Ras-GTP2) to produce phosphatidylinositol-3,4,5-trisphosphate P3). PtdInsP3 acts as a second messenger activating AKT-dependent and AKT-independent signaling pathways that regulate diverse cellular processes including cell proliferation, survival, motility, and metabolism [The most well characterized transforming pathways downstream of Ras are the MAPK and PI3K effector pathways. Activation of MAPK signaling is initiated by Ras-GTP binding of RAF kinases that results in localization to the plasma membrane and activation of their serine/threonine kinase activity , 11. Acttabolism .The failure to develop effective pharmacologic inhibitors of Ras oncoproteins has led many to conclude that Ras is unruggable . The inhKRAS-driven transformation of epithelial cells [NRAS(V12) and Mll-AF9-driven mouse model (tNM AML) [There is mounting evidence that the Ras-like  proteins are critical mediators of Ras-driven transformation, proliferation, migration, and survival of epithelial cancers . For exaal cells . WhethertNM AML) , human ANRAS oncogene expression in the tNM AML model [NRAS(V12) oncogene from a tetracycline-response element (TRE) promoter, and NRAS(V12) transcription can be suppressed by administering the tetracycline analog doxycycline (Dox). To confirm the NRAS(V12) dependence of tNM AML cells we first transplanted tNM AML cells into SCID Beige recipient mice, and monitored their blood leukocyte counts. After treatment with doxycycline, NRAS protein expression was undetectable in splenocytes from leukemic mice by Western blotting at 72 hours  expression resulted in normalization of leukocyte counts within five days of doxycycline treatment  after a 48 hour ex vivo treatment of tNM AML cells with doxycycline  suppression on MAPK, PI3K, and RALB signaling pathways downstream of Ras, we measured the levels of phosphorylated ERK1/2 (pERK1/2), 4E-BP1 (p4E-BP1), and TBK1 (pTBK1) in tNM AML cells. Suppression of NRAS(V12) expression decreased the levels of pERK1/2, p4E-BP1, and pTBK1  expression on cell proliferation and apoptosis we evaluated cell cycle profiles and levels of cleaved Caspase 3 (CASP3) by flow cytometry. Suppression of NRAS(V12) expression led to apoptotic cell death but did not block cell cycle progression  alone or the PI3K/mTOR inhibitor BEZ235  alone had no significant anti-leukemic effect in vivo despite evidence of inhibition of the target pathways (data not shown). Furthermore, treatment with the combination of AZD6244 and BEZ235 resulted in stabilization of leukocyte counts during the treatment period  and RAS wild-type  human AML cell lines. In human AML cells, inhibiting MAPK signaling with AZD6244 had a modest effect on viable AML cell numbers  in the tNM AML model supports a critical role of Ras signaling to support AML survival. Surprisingly, inhibition of the major transforming pathways downstream of Ras, MAPK and PI3K, alone or in combination did not reproduce the pro-apoptotic effects of disruption of oncogenic Ras in this model or in human AML cells. While incomplete or transient inhibition of the target pathways could contribute to the differences observed, these findings are consistent with previous work in a variety of AML models [Many common mutations in AML lead to activation of Ras signaling, making Ras and its effectors an attractive therapeutic target. A major obstacle to Ras-targeted therapy for AML is the inherent difficulty in targeting Ras directly. Although targeting Ras effector pathways is a more tenable approach, the optimal targets downstream of Ras for AML are not known. The effects of genetic disruption of L models –20. FurtL models . TargetiL models . SeveralL models , 26, butL models , combineNRAS oncogene expression on AML cells, we hypothesized that RALB signaling might be a key survival pathway. Murine knockout studies have shown an essential role for either RALA or RALB in KRAS driven non-small cell lung cancer cell proliferation [KRAS-driven transformation of epithelial cells through it's effects on BCL2 family protein expression [Based on the failure of inhibiting MAPK and/or PI3K signaling to reproduce the effects of direct suppression of feration . Furtherpression . Consistpression . FurtherOverall, our results provide new insight into the effects of specific Ras signaling pathways on AML proliferation and survival, and have uncovered a novel role for RALB signaling in AML survival. This work not only refines our understanding of how specific Ras effectors promote proliferation (PI3K and MAPK) and survival (RALB) of AML cells, but also provides a biologic basis to guide the rational development of Ras-targeted treatment approaches for AML patients.Protein lysates were run on 10% PAGE gels and transferred to a PVDF using the NuPAGE and iBlot systems (Life Technologies). Blots were blocked and stained according to antibody manufacturer's recommendations. Blots were developed using the SuperSignal West Pico ECL kit (Thermo Fisher) and visualized and signals were quantified using the LI-COR imaging system (LI-COR Biosciences). Actin goat polyclonal (sc-1616), NRAS mouse monoclonal (sc-31), and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology; RALB rabbit polyclonal (3523), α-Tubulin rabbit monoclonal (2144), and GAPDH rabbit monoclonal (2118) antibodies were purchased from Cell Signaling Technologies; and BCL2 mouse monoclonal (610538) antibody was purchased from BD Biosciences.For staining of intracellular antigens, cells were fixed with 2% paraformaldehyde (Electron Microscopy Sciences) and permeabilized with 90% methanol (Sigma). Cells were stained according to manufacturer's recommendations. Cleaved caspase 3  PE, cleaved PARP  Alexa Fluor 647, phospho-4E-BP1  Alexa Fluor 647, phospho-AKT  Alexa Fluor 488, phospho-p44/p42 MAPK  PE, and phospho-TBK1  PE were purchased from Cell Signaling Technologies; and phospho-TBK1  Alexa Fluor 488 was purchased from BD Biosciences. Cell cycle analysis was performed using the PI/RNase Staining Buffer (BD Pharmingen). Cells were analyzed on an LSR II or Fortessa digital flow cytometer (BD Biosciences), and cell sorting was performed on a FACS Aria II (BD Biociences) at the University of Minnesota Flow Cytometry Resource.Cell lines were originally obtained from ATCC or DSMZ, maintained under standard cell culture conditions, and authenticated by STR analysis at the University of Arizona Genomics Core. RALB-targeted  and non-silencing (NS) control GIPZ shRNA plasmids were obtained from Dharmacon. RALB(S28N) and BFP control expression vectors were generated with the Gateway cloning system (Invitrogen) using a previously described backbone , by repltNM AML cells were cultured in RPMI-1640 medium (Lonza) with 10% fetal bovine serum  with or without 1 μg/mL doxycycline (Sigma) for 48 hours and then plated in IMDM medium (Lonza) with 30% fetal bovine serum , 1.275% methylcellulose (R&D Systems), and 2 ng/mL murine GM-CSF (R&D Systems). Human cell lines were treated with pathway inhibitors for 24 hours in standard culture medium and then harvested and plated in MethoCult H4034 Optimum CFC Medium (Stem Cell Technologies) according to manufacturer's recommendations. Colonies were scored after 7–14 days on an inverted microscope.® Aqueous Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer's recommendations. Pathway inhibitors stocks were prepared in dimethyl sulfoxide , and diluted in cell growth medium to their final concentrations. Half-maximal inhibitory concentrations (IC50) and combination indices were calculated using CalcuSyn 2.0 (BioSoft). Inhibitor combinations were considered synergistic if CI < 0.8, additive if CI 0.8–1.2, or antagonistic if CI > 1.2.Relative viable cell numbers were determined using the CellTiter 966 tNM AML cells were injected via tail vein into 6–10 week old SCID Beige (Charles River) recipient mice without any preconditioning or FVB × C57BL/6 F1 (Jackson Labs) recipient mice after 500 cGy irradiation. Peripheral blood was obtained by retro-orbital blood sampling, and white blood cell (WBC) counts were monitored using a Hemavet 950 (Drew Scientific). Doxycycline treated mice were given a single intraperitoneal dose of 4 mg followed by 5 mg/mL in the drinking water. AZD6244 and BEZ235  were diluted in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 80 and administered by oral gavage.All animal studies were approved by the U of M Institutional Animal Care and Use Committee. Briefly, 2 × 10


Ars […] de statica medicina (On static medicine). This is a work composed of aphorisms that present the practical results of a series of weighing procedures, rather than theoretical observations. De statica medicina is the result of a large number of test series that Sanctorius carried out over many years with the weighing chair he constructed himself in order to quantify the so-called perspiratio insensibilis, an insensible perspiration of the human body. Through his weighing experiments, Sanctorius introduced the idea of quantitative research into physiology. Although historical accounts ascribe an important role to Sanctorius as the founder of a new medical science, up until now the design of his weighing chair and the method of measurement have not been closely analysed. The aim of this paper is to close this gap. Through a collaboration between the Max Planck Institute for the History of Science and the Technical University of Berlin , Sanctorius’s weighing chair was reconstructed and experiments carried out with it. This opened new perspectives on Sanctorius’s work and led to a reconsideration of the function and purpose of his weighing chair. With his static medicine, Sanctorius repurposed an old instrument. The replication of the weighing chair and the repetition of the experiments demonstrate that this novel application of scales posed some challenges for the mechanical design of the instrument. We recognized that the instrument fulfilled different functions that might in turn have affected its design, precision, and the measuring method applied. Although in the end we could not clarify how Sanctorius actually conducted his measurements, we were nevertheless able to develop an understanding of Sanctorius’s mechanical and practical knowledge that would not have been possible for us to develop solely on the basis of the written sources.In 1614, the physician Sanctorius Sanctorius (1561–1636) published his most famous work entitled  The use of balances is very normal to us today. We use kitchen scales to bake cakes, weigh birthday parcels in order to calculate the postal charges, and try desperately not to exceed the weight limits for our luggage before heading to the airport. To keep track of our own weight, most of us own bathroom scales, which have become more and more precise and can now even determine the percentage of body fat or muscle mass in a human body.In effect, the balance is one of the oldest measuring instruments. Most probably, it was invented during the Neolithic period, when commerce and trade routes began to develop and the first towns were established , we reconstructed the weighing chair of Sanctorius and conducted a series of experiments with it, partly undertaken in the framework of a seminar at the History of Science Department at the Technical University Berlin.In what follows, I will present the current state of our results in three steps. First, I will analyze the primary sources on Sanctorius and his weighing chair in order to understand the instrument and the weighing process; I will also describe their reception and interpretation. Second, I will give an account of our reconstruction of and experiments with the Sanctorian weighing chair. Finally, I will summarize the conclusions we have drawn from these two lines of research. In practice, these elements were of course not separate from each other but rather closely intertwined and often mutually stimulating. The continuous rereading of the sources, combined with the knowledge that we gradually gained by reconstructing and conducting experiments with the Sanctorian weighing chair, led us to a new understanding of Sanctorius’s static medicine.Ars […] de statica medicina in Venice  that was published in 1625—more than ten years after the De statica medicina:perspiratio insensibilis of our bodies occurs daily: which, if not rightly weighed, renders medicine altogether vain. For nearly all bad illnesses usually originate from a smaller or larger perspiration than is proper. Secondly, sitting in this chair and easily eating in between, we observe when we reach the due quantity of food and drink in excess of which or in shortage of which we are injured. The chair is arranged as it appears in the figure , which constitute the main determinants of health and disease in traditional dietetic medicine  air, (2) food and drink, (3) sleep and wakefulness, (4) motion and rest, (5) evacuation and repletion, (6) passions of the mind. Management of the patient’s regimen  was for centuries the physician’s most important task , De Cibo & potu (food and drink), De Somno & vigilia (sleep and wakefulness), De Exercitio & quiete (exercise and rest), De Venere (coitus), De Animi affectibus (affections of the mind). These are sections II to VII. Section I is entitled De ponderatione insensibilis perspirationis and deals with the method of weighing insensible perspiration  which suppose to be twenty Ounces: Then chuse such a settled Quantity of Food, and keep to such a use of the Non naturals, as will bring the Quantity perspired to a Mean between fifty and twenty Ounces, which is thirty five Ounces; and by this means may a Person be brought to such a perfect Standard of Health as will last to a Hundred Years  have felt the need to highlight that there was only a chair—and not a table or a bed, as others claim—hanging from the steelyard  hanging on the short arm. When the two arms are balanced in a horizontal position about the pivot, the weight of the load is indicated by the position of the counterweight on the graduated arm. Thus, the weight can be either read directly from the graduation marks or calculated according to the law of the lever . In this method, the counterweight does not have to be moved for every individual to reach the starting point. This enables balancing the weighing chair without climbing up to the ceiling over the dining room.21descent of the chair. Thus, he used the weighing chair not only to observe weight loss but weight gain as well. Where this second marking has to be—meaning the mark for the quantity of food and drink Sanctorius advises individuals to ingest—remains vague to the modern reader. Sanctorius not only connects it with the amount of excreted insensible perspiration, but also refers to the six non-naturals, whereby he includes a variety of parameters that influence the quantity of food and drink that an individual person should ingest. This leads to the conclusion that Sanctorius based the position of the second marking on contemporary dietetic knowledge and the experiences he gained during the weighing procedures.In addition to the starting point, there has to be at least one other marking at the bottom of the chair. As mentioned above, Sanctorius refers in his description of the weighing chair to a certain measure that is set before the weighing starts and indicates to the sitter when he has ingested the sufficient amount of food and drink. Sanctorius explicitly states that the quantity of ingested food and drink is indicated by the So when we tried to define this second marking in our experiments with the reconstruction, we did not deal with an exact quantity but rather attempted to determine how a certain quantity (the second marking) could generally be determined for different individuals using the chair. Here again, different possibilities can be found through different methods of determining the starting point. If we balance the beam by moving the counterweight hanging from the steelyard above the ceiling and we do this for every individual with regard to their weight, the descent of the chair is proportional to the weight of the load. Therefore, a universal marking could be attached to the bottom of the chair, indicating when each individual person has ingested the sufficient amount of food and drink. Another option is to determine the weight of a person by the steelyard in the common way and then use the same method again to set a desired weight for the food and drink that a person should ingest. The counterweight could be moved to such a position that once the previously set measure is reached the chair not only drops down but touches the ground. Using the ground floor in this way as an indicator of when the desired amount of food and drink has been ingested is of course also possible for the method mentioned before, instead of setting a mark close to the bottom of the chair. If we use the other option and add weights to the person sitting on the chair to balance the beam of the steelyard, the amount of food ingested can be indicated by a graduated scale attached to the bottom of the chair. Here the initial weight of the load is the same for everyone using the chair. Thus, the descent of the chair after a meal is not proportional to the weight of the individual person. The addition or removal of weights to or from the chair might have enabled Sanctorius to identify exact quantities not only above the ceiling by looking at the position of the movable weight hanging from the beam of the steelyard, but also by looking at the bottom of the chair.As soon as we started to include the descent of the chair in our procedures and tested its possible function as an indicator of changes in weight, flaws in our reconstruction came to light. It turned out that the chair was very unsteady and sensitive to any kind of movement. Thus, the various persons using the chair not only had to keep still during the measuring process, but also had to adopt an identical seating position. To prevent the chair from rotating to one side, we replaced the rope that suspended the chair in our first version of the prototype with a steel chain that we attached to the chair with the help of a U-bolt see Fig. . AdditioOur experiences showed that the suspension of the chair to the beam, as depicted in the original illustration, is prone to rotation and prevents a precise reading of measurements by means of the chair’s descent. However, Sanctorius was well aware of this difficulty, as he states in the description of the weighing chair: “the chair remains […] stable in such a way that it cannot be easily moved; […] , Sanctorius states that up to 16 ounces of urine are usually expelled in one night. In several other aphorisms, especially of the third section, Food and Drink, he gives quantities of six, twelve, 14, 18, and 22 ounces. He writes for example: “Very nourishing Meats, excepting Mutton [sic] between the time of Supper and Dinner, do not Perspire above Eighteen Ounces  to measure certain parameters, as for example in the last section, De Animi affectibus (affections of the mind). Here, Sanctorius demonstrates how rage, joy, fear, melancholia, and consolation affect perspiration: “Amongst the Affections of the Mind, those of Anger and Joy, make Persons lighter, those of Fear and Sorrow, more heavy; and the other Affections operate in Proportion to their Participation of these , is far easier to apply to the weighing procedures. The quantities of ingested food and drink can be controlled and monitored relatively easily. This might also be the reason why Sanctorius specifies many precise quantities in this section.Interestingly, Sanctorius only specifies precise quantities in the first four sections of the De statica medicina, but to develop a general understanding of their method. Our experiments showed that our reconstruction needs to be further adapted to allow for a full analysis of Sanctorius’s weighing procedures. Systematic long-term measurements, including all of the parameters that Sanctorius mentions, are planned. However, in this pilot phase we tried to reenact the weighing procedures as much as possible. We created tables for every person that used the chair to record their age and mood, the time and date, and the weather before the measurements were taken. Moreover, they had to indicate whether they moved or rested before the weighing took place, whether they slept or were awake, and whether they consumed food and drink. After the measurements had been performed, they had to calculate how much food they ingested, how much they excreted insensibly, and how much they excreted through feces. Thus, we had to weigh them at regular intervals during the duration of the experiments, immediately before and after eating, and immediately before and after going to the toilet. In keeping with our interpretation of Sanctorius’s description of the device (see fn. 12), we did not allow a person to eat whilst in the chair. At this stage, the calculations were intended to give us a general idea of how Sanctorius’s weighing practice might have looked; they did not provide reliable data to develop a scale or to verify Sanctorius’s measurements. Nevertheless, these experiments made us understand how difficult it is to include all the various parameters in the measurements and to consider them in conjunction with one another. This is a difficulty that Sanctorius faced himself. In his aphorisms, he only ever determined a selection of the interactions between the many parameters and their impact on the physiology of human beings. There is no coherent list of parameters that he takes into account in every measurement. Hence, apart from the mechanical challenges of the weighing chair, a comprehensive imitation of Sanctorius’s procedures requires careful preparation and a close reading of the aphorisms of the De statica medicina. It demands a high level of self-discipline and a regular and uniform lifestyle, always within reach of the weighing chair.25The aim of our experiments was not to verify the exact results that Sanctorius presents in the With his weighing chair, Sanctorius repurposed an old instrument. Our reassessment of the original source materials based on the experience gained through reconstructing the Sanctorian chair and replicating the weighing experiments taught us how this novel application of the steelyard raises challenges for the mechanical design of the instrument. It also widened our perspective to the great variety of its applications. Different measuring methods can be applied that directly affect the design, the functioning, and the precision of the weighing chair. Although our research does not allow us to unambiguously define the measuring method Sanctorius used, it has shown that this method is not as self-evident as was commonly assumed and that it warrants further investigation. Systematic long-term measurements with a further-adapted version of the reconstruction have to be undertaken.On the basis of our research, we can assume that Sanctorius most likely used some variation of the measuring methods mentioned above. He used both the steelyard concealed behind the ceiling and at least two reference points attached to the bottom of the chair. Even though the original illustration of the weighing chair gives no clear indication of a scale at the bottom of the chair or on the wood panel behind it, scaling would have been necessary to these two reference points. In short, Sanctorius had to translate weight into a distance. He thus worked with proportions as well as with exact quantities. Whether the pointers or pegs at the bottom of the chair served as indicators for these reference points, whether they were meant to stabilize the chair, or whether they fulfilled both functions requires further investigation.De statica medicina and the description of the Sanctorian chair imply that the instrument had two functions. On the one hand, it was used as a research tool to monitor variations in the production of perspiratio insensibilis; on the other, it helped to determine and maintain an ideal body weight. The measuring methods might have varied in correspondence with these two functions. Based on our experiences with the reconstruction, it seems likely that Sanctorius used the steelyard in the traditional way, especially in the initial phase of his experiments, when he tried to define the healthy quantity of insensible perspiration. As soon as he managed to stabilize this quantity, he could determine the ideal body weight for different individuals and determine the healthy amount of food and drink that they should ingest. To this purpose, he might have used the descent of the chair as an indication of changes in weight, as described in the commentary on Avicenna. This would have enabled individuals, even laymen, to use the chair on their own, without the need of an assistant to move the counterweight along the longer arm of the weighing chair. In this regard, the weighing chair would not have been meant for use by multiple individuals, but only by one person; the beam of the steelyard would therefore be balanced only once, for that person’s respective weight. This fits with Sanctorius’s suggestion that the beam of the steelyard be hidden above the ceiling to avoid the astonishment of guests, to whom the weighing device would look ridiculous. It implies that Sanctorius may have conceived of the chair for a larger public to regulate their eating habits.26The aphorisms of the De statica medicina. This could reflect the development of his research during these years. After beginning with the aim to determine the quantity of the perspiratio insensibilis within the frame of contemporary dietetic medicine, he might have realized that the chair not only helped the physician to monitor changes in weight and, on this basis, to issue rules of health, but also offered the opportunity to find and maintain an ideal weight. In order to make the chair accessible to laymen, he might have adapted the design and measuring method with regard to this newly discovered function and published both in his commentary on Avicenna.27In this context, it is important to keep in mind that Sanctorius published the description and illustration of the Sanctorian chair eleven years after the Over the course of the eighteenth century, the two functions of the Sanctorian chair were hotly debated Dacome : 475. WhI thank Friedrich Steinle, Hans-Liudger Dienel, and Jürgen Renn, who have rendered the collaboration between the Max Planck Institute for the History of Science and the Institute of Vocational Education and Work Studies (TU Berlin) possible. Friedrich Steinle provided invaluable comments that greatly improved the manuscript. I would like to thank Katharina Wegener and Volker Klohe, who supported me in the process of the replication; without their help, it would have been impossible to realize this project. I am also immensely grateful to Jochen Büttner, who provided insight and expertise that greatly assisted the research. I would also like to thank Lindy Divarci, Zachary Gresham, and Lindsay Parkhowell for their editorial work. Last but not least, I sincerely thank Matteo Valleriani for his ongoing support throughout the whole project and for his precious advice, comments, and critiques.


Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions.The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [ The translationally controlled tumor protein (TCTP) was initially reported as a tumor-associated and growth-related protein (reviewed in ). Its hoMounting evidence has indicated the potential for the regulatory roles of TCTP in the brain functions. TCTP mRNA is expressed in many areas of the adult human brain , and sig3H]dopamine release and that the TCTP-triggered dopamine release from PC12 cells is mediated by Ca2+-independent, phospholipase A2 (PLA2)-involved pathways.Here, motivated by the suggestive implications of TCTP in neurons and its localization in neuronal systems, we investigated the role of TCTP in the neurotransmitter release from a neurosecretory cell type, pheochromocytoma (PC12) cells. Our present study reports that TCTP enhances both basal and depolarization-evoked i). Fura-2-acetoxymethyl ester (AM), a widely used membrane permeable Ca2+ indicator, is known to be hydrolyzed by intracellular esterases, and then trapped within the cytoplasm. Fura-2-AM-loaded PC12 cells were treated with MrTCTP or KCl in the Ca2+-free or Ca2+-containing KR buffer. MrTCTP evoked a gradual and sustained increase in [Ca2+]i in the Ca2+-containing buffer condition but not in the absence of extracellular Ca2+ , as shown in 2+ current evoked by KCl was inhibited (data not shown). This finding suggests that unlike KCl, voltage-gated L-type Ca2+ channels are not involved in MrTCTP-induced Ca2+ current.It is well-known that the neurotransmitter release stimulated by depolarization is Calar Ca2+ A, simila [Ca2+]i B. Removaondition C. It hasocytosis ,14. The 2+]i increase was due to the mobilization of Ca2+ from intracellular stores by utilizing dantrolene, which blocks Ca2+ release from the endoplasmic reticulum (ER). The rTCTP-evoked cytosolic Ca2+ current was also not affected by dantrolene (10 µM), as shown in 2+]i was due to a Ca2+ influx from an extracellular source rather than via voltage-gated L-type Ca2+ channels, and that rTCTP does not mobilize Ca2+ from intracellular stores. Thapsigargin, an inhibitor of Ca2+-ATPase in the membranes of ER Ca2+ stores, is generally used to inhibit refilling of Ca2+ stores and subsequently depleting Ca2+ from Ca2+ stores in various cells i, confirming that intracellular Ca2+ stores are releasable under our experimental conditions. When 50 µg MrTCTP were added in the medium that contained both Ca2+ and thapsigargin, further rise in [Ca2+]i was not produced by rTCTP  on dopamine release from PC12 cells were examined. Unexpectedly, rTCTP stimulated dopamine release even in the absence of extracellular Ca2+ by a similar extent to that in the presence of extracellular Ca2+. In contrast, both bradykinin (1 µM) and KCl (50 mM) stimulated dopamine release only in the Ca2+-containing media, and did not induce dopamine release in the absence of extracellular Ca2+ (Ca2+-free media + EDTA (ethylenediaminetetraacetic acid)) (2+-independence of the rTCTP-evoked dopamine release was further confirmed by the observations that BAPTA-AM (10 and 50 µM), nifedipine (2 µM) and dantrolene (10 µM) did not significantly inhibit rTCTP-evoked dopamine release either in the presence or in the absence of extracellular Ca2+ ) A. The Calar Ca2+ B–D, althg buffer . These d2+-dependent and Ca2+-independent mechanisms Dopamine (125 µCi/mmol) was purchased from Amersham Bioscience Corp.  and the Scintillation cocktails (ScintiSafeTM Econo2) were from Thermo Fisher Scientific . Bromoenol lactone (BEL), arachidonyl trifluoromethyl ketone (AACOCF3), and 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3-phosphorylcholine (TEA-PC) were from Biomol Research Laboratories . Nickel-nitrilotriacetic acid (Ni-NTA) resins were from Qiagen . Fura-2 acetoxymetyl ester (Fura-2-AM) was purchased from Molecular Probes . Pargyline, desipramine, bradykinin, nifedipine, dantrolene, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), isopropylthiogalactoside (IPTG), and other chemicals and reagents were purchased from Sigma Aldrich  or BD Difco . Stock solutions for Fura-2-AM, BAPTA-AM, BEL, AACOCF3, TEA-PC, dantrolene, and bradykinin were prepared by dissolving the compounds in dimethyl sulfoxide (DMSO), and then further diluted in serum-free media; final DMSO concentrations did not exceed 0.2% (v/v). RPMI 1640, penicillin/streptomycin, horse serum, and fetal bovine serum (FBS) were obtained from GIBCO . dopamine (0.5 µCi/mL) for 3 h at 37 °C. The medium was removed and the cells were washed three times with PBS containing 1 mM ascorbic acid. To measure both basal and depolarized dopamine release, cells treated with various indicated reagents including rTCTPs were incubated at 37 °C for 20 min in 1 mL Krebs-Ringer-HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer  and the buffer containing 50 mM KCl, respectively. EGTA (0.5 mM) was added to the Ca2+-free (without CaCl2) media. Samples were centrifuged for 10 min at 1000× g at 4 °C to remove floating cells, and the supernatant was harvested and 0.2 mL was measured for released radioactivity. For total radioactivity, the cells were washed with PBS and solubilized in 1 mL of 0.5 N NaOH, and 0.45 mL was measured for radioactivity. PC12 cells were grown in 24-well dishes in serum-containing medium for 24 h at 37 °C. The medium was removed and cells were loaded with fresh serum-free medium containing i were determined by monitoring Fura-2 fluorescence in a Shimadzu RF-5301 spectrofluorometer  by the dual wavelength method of Grynkiewicz et al. i only under the Ca2+-containing condition. The current study strongly suggests that the rTCTP-induced neurotransmitter release from PC12 cells is mainly regulated by Ca2+-independent pathways where iPLA2 may be involved. Further studies are needed to verify the TCTP’s role in the dopamine release using the in vivo experimental system or primary cultured rat neuronal cells. We found a novel neuronal function of TCTP in stimulating dopamine release in both basal and high K