SLC22A3 encodes an organic cation transport protein, which is critical for drug transportation and cellular detoxification. SLC22A3 is significantly downregulated and associated with tumor progression and worse prognosis in a variety of solid tumors. However, there are no data available regarding the role of SLC22 in AML. This study aimed to explore the regulatory mechanism of DNA methylation on SLC22A3 expression, as well as its clinical significance in AML prognosis.We screened out several hypermethylated solute carrier (SLC) family genes in acute myeloid leukemia by reduced representation bisulfite sequencing. SLC22A3 was identified as the sole prognosis-associated gene among SLCs based on TCGA and Beat AML databases. Bone marrow mononuclear cells (BMMNCs) from AML, MDS patients, and healthy donors were enrolled in this study. SLC22A3 methylation was significantly increased in AML compared with controls and MDS patients; meanwhile, the expression level of SLC22A3 was decreased. SLC22A3 hypermethylation presented an obvious association with some specific clinical characteristics and affected the survival time of AML patients as an independent risk indicator. SLC22A3 expression changed regularly as the disease complete remissions and relapses. Demethylation drug 5-aza-2′-deoxycytidine (DAC) activated transcription and increased mRNA expression of SLC22A3 in leukemia cell lines and AML fresh BMMNCs. Knockdown of SLC22A3 in leukemia cells enhanced cell proliferation and suppressed cell apoptosis. Data from public programs were used for auxiliary screening of probable molecular mechanisms of SLC22A3 in the antileukemia effect.SLC22A3 may be indicators of poor prognosis in AML. Methylation-silenced SLC22A3 expression may have potential guiding significance on antileukemia effect of DAC.Our results showed that increased methylation and decreased expression of The online version contains supplementary material available at 10.1186/s13148-022-01373-w. Recent decades have witnessed an increasing trend of leukemia incidence and mortality in china and worldwide –3. AML iSLC22A3 and cancers, including tumorigenesis, tumor invasion and metastasis, uptake and metabolism of antineoplastic drugs, and disease prognosis , 93 newly diagnosed MDS patients, and 45 healthy donors were enrolled in this study. BMMNCs were isolated by Ficoll density gradient centrifugation. Our research was approved by the Institutional Ethics Committee of the Affiliated People’s Hospital of Jiangsu University, and each individual provided signed informed consents for their participation. The diagnosis and classification of MDS and AML patients were based on the French–American–British (FAB) and the 2016 World Health Organization (WHO) criteria .A series of clinic-hematological profiles and auxiliary examination results of patients were enrolled in this study for clinical correlation research. Hematologic laboratory results, including morphologic identification of bone marrow aspirate, cytogenetic analysis, immune-phenotypic feature, and molecular testing were valuable for the diagnosis and prognosis of hematopoietic malignancies. These indexes were also applied to follow-up study after induction and consolidation chemotherapy , 25. CytSLC22A3 expression was detected by RT-qPCR using the SYBR Premix Ex Taq II  and was calculated by relative expression level (2 − ΔΔCt) with ABL as internal reference [SLC22A3 (71 bp), 5′-CCACCATCGTCAGCGAGT-3′ (forward), 5′-CAGGATGGCTTGGGTGAG-3′ (reverse); internal reference ABL (118 bp), 5′-TCCTCCAGCTGTTATCTGGAAGA-3′ (forward), 5′-TCCAACGAGCGGCTTCAC-3′ (reverse). The appropriate primers and personalized RT-qPCR reaction temperature partly different from the manufacturer’s template were verified by gel electrophoresis and Sanger sequencing of PCR products, 60 °C for 30 s in annealing temperature and 82 °C for 30 s in collected fluorescence temperature.Total RNA was isolated as per the TRIzol reagent instruction  from each BMMNCs sample and was transcriptionally reversed into cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit . RNA concentration and quality were assessed with a NanoDrop 2000 . eference . PrimersGenomic DNA was isolated from pretreated BMMNCs samples by Genomic DNA Purification Kit  and then modified using CpGenome DNA Modification Kit . Methylation sites in genomic DNA were exposed after the bisulfite conversion and can be identified by targeted primers .SLC22A3 promoter were validated by targeted bisulfite sequencing (methyl target) sequencing , a multiple targeted CpG methylation analysis by next-generation sequencing [SLC22A3 , 5′-GGGATTAAAAGGAGTTTCGC -3′ (forward), 5′-CACTCGCCCTAACGCTATAC -3′ (reverse); unmethylated SLC22A3 , 5′-GTAGGGATTAAAAGGAGTTTTGT -3′ (forward), 5′-CCTCACTCACCCTAACACTATAC -3′ (reverse); internal reference ALU (110 bp), 5′-TTAGGTATAGTGGTTTATATTTGTAATTTTAGTA-3′ (forward), 5′-ATTAACTAAACTAATCTTAAACTCCTAACCTCA-3′ (reverse). The relative methylation level of SLC22A3 was calculated by the formula: NM/U-SLC22A3 = 2M/U−SLC22A3(control−sample)ΔCT  ÷ 2ALU(control−sample)ΔCT (2−ΔΔCT). RQ-MSP reaction systems were personalized in annealing and collected fluorescence temperatures, 66 °C (M) or 63 °C (U) for 30 s and 80 °C for 30 s, respectively.Based on the CpG sites of the genomic promoter region, we designed the primers and check them for feasibility and specificity. The bisulfite convert ratio and methylation levels of 27 CpG sites located at quencing . The 20μ◦C humidified atmosphere containing 5% CO2.The human leukemia cell lines K562 (FAB-M6), HL-60 (FAB-M2), THP-1 (FAB-M5), U937 (FAB-M5), SKM-1 (MDS-derived AML M5), and MV4-11 (FAB-M5) were purchased from the American Type Culture Collection  and gifted from other laboratories. Cells were cultured in freshly prepared RPMI 1640 medium  supplemented with 10% fetal calf serum  and 1% penicillin–streptomycin , surrounded by a 37Fresh mononuclear cells from the bone marrow of four newly diagnosed untreated AML patients  were separated through density gradient centrifugation using a PBMC separation tube  and suspended in Dulbecco's phosphate-buffered saline  for twice, according to the specification protocol. After cultured in the same medium and environment as cell lines for 48 h, all the suspension cells were collected for further culture, while mesenchymal stem cells which grew by static adherence were isolated.SLC22A3 (siSLC22A3) and its related negative control (siNC) were designed and synthesized by GenePharma . Cell transfection was performed using EntransterTM-R4000 .Gene silencing induced by small interfering RNAs (siRNAs) was performed in HL60, K562, and AML fresh BMMNCs. siRNAs against 5 cells/ml) and AML fresh BMMNCs (1 × 105 cells/ml) were seeded onto a 6-well plate with normal substrate environment. After culturing for 0, 24, 48, and 72 h, cell proliferation status was counted on the counting board three times.Cell lines (1.5 × 105 cells/ml) were seeded onto a 6-well plate in RPMI 1640 medium without fetal calf serum for 72 h. Cell apoptosis rate was detected by flow cytometry  using Annexin V-PE/7-AAD apoptosis detection kits . Viable cells were counted using 0.4% trypan blue staining . Each experiment was repeated three times.Cells  sensitivity experiment. Normal growing cells lines and AML fresh BMMNCs (1 × 105 cells/ml) were treated by DAC at a final concentration of 0 μM, 0.1 μM, 1 μM, and 10 μM for 72 h, respectively. The expression of SLC22A3 in treated cells at each concentration was determined by RT-qPCR. After treated by DAC at a final concentration of 1 μM for 24 h, cell apoptosis of K562 siNC/siSLC22A3 was detected by flow cytometry.According to Our laboratory detected many differential methylation genes within four normal donors and four pairs of MDS-sAML (secondary AML) patients through RRBS . The rawhttp://www.cbioportal.org) and DiseaseMeth version 2.0 (http://bio-bigdata.hrbmu.edu.cn/diseasemeth/index.html) [http://www.ncbi.nlm.nih.gov/geo/; GSE63409) were used for gene screening. Genomic sequencing and clinical data of 558 cases from Beat AML program were downloaded via cBioPortal  [Gene expression (RNA Seq V2 RSEM), methylation (HM450), and clinical information of 200 adults with de novo AML (NEJM 2013) from the Cancer Genome Atlas (TCGA) dataset were downloaded via cBioPortal (ex.html) . Genome-tal.org) . Differetal.org) .U test and Paired T test) were performed to compare the differences of continuous variables. The Pearson chi-square analysis/Fisher exact test was conducted to analyze the diversities of categorical variables, while Spearman correlation test was used to evaluate the correlation between genes expression and methylation. The ROC curve and area under the ROC curve (AUC) were used for assessing discriminative capacity of SLC22A3 methylation between patients and controls. Kaplan–Meier and Cox regression (univariate and multivariate) analyses were carried out to evaluate the impact of SLC22A3 on survival. Statistical significance was set at P < 0.05, and each test was two-sided.SPSS 22.0 software package and GraphPad Prism 5 were applied to statistical analyses. Student's t test  and disease-free survival (DFS) in both non-acute promyelocytic leukemia (APL) AML (non-APL AML) (P = 0.008 and 0.002) and cytogenetically normal AML (CN-AML) patients  . The mean bait coverage attached 1694 × , and Q30 was 75.56% [SLC22A3 was significantly increased in MDS and AML patients compared with controls (P < 0.001 and < 0.001), as well as in AML compared with MDS  and AML (n = 153) samples by RQ-MSP, a more convenient method. Primers for RQ-MSP and targeted sequencing were designed most overlapped in ampliconic sequence. In addition, RQ-MSP results showed a high positive correlation with that in the targeted bisulfite sequencing as confirmed , as well as in non-APL AML (n = 107) and CN AML (n = 58) vs. controls, respectively (P < 0.001 and = 0.002). However, we did not find a distinct variation between controls and MDS specimens.To further confirm the pattern of SLC22A3 methylation could be helpful to distinguish AML from controls  based on the cutoff value of 0.042 (according to Youden index of ROC curve), which can also put almost all healthy donors in hypomethylation set. SLC22A3 hypermethylation was associated with lower platelets and higher CEBPA mutation rate in non-APL AML patients (P = 0.020 and 0.006), as well as higher mutation rate of N/K-RAS in CN-AML   Table . HoweverSLC22A3 methylation affect the life span expectancy of MDS and AML patients. We screened AML patients treated with 3 days of an anthracycline and 7 days of cytarabine (“3 + 7” regimens). Kaplan–Meier analysis indicated tendencies of shorter OS and leukemia-free survival (LFS) in CN-AML patients with SLC22A3 hypermethylation  and/or clinically recognized related to AML into multivariate analysis. In non-APL AML cohort, SLC22A3 hypermethylation was an independent risk indicator for shorter OS and LFS, respectively  and AML (n = 89) were used to assess SLC22A3 expression level by RT-qPCR. Our results showed that SLC22A3 expression was downregulated in AML compared with controls and MDS .Since SLC22A3 expression in AML, we created conditions of SLC22A3 silencing in HL60 and K562 cells, as well as fresh BMMNCs from two AML patients. We transfected siSLC22A3 and its related siNC into these tool cells, and obtained remarkable silencing effect  of CEBPA mutations separately because of the limited mutation cases. We observed no correlation between SLC22A3 expression and CEBPA biallelic mutations based on Beat AML databases. Considering of the prognosis differences between two types of CEBPA mutations, the causal relationship between SLC22A3 hypermethylation and CEBPA mutation and its clinical significance in AML remain to be clarified.The transcription factor CCAAT enhancer binding protein alpha (CEBPA) is a myeloid transcription factor. Ley et al. confirmed that mutations in CEBPA and other myeloid transcription factor genes, such as DNMT3A, NPM1, IDH1/2 and RUNX1, were common in AML and suggested that these mutations had functions related to the pathogenesis of AML . ResultsSLC22A3 methylation conferred susceptibility to esophageal squamous cell carcinoma [Promoter methylation plays an important role in SLC22A3 expression. Chen et al. confirmed that methylation in the SLC22A3 promoter region could explain the low expression level of SLC22A3 in high-Gleason grade prostate cancer, which may be related to the progression of prostate cancer. In SLC22A3-negative HCT116, SLC22A3 mRNA levels were significantly reactivated with increased dose of 5′ -AZADC (a demethylating agent) . Xiong earcinoma .SLC22A3 mRNA expression of two leukemic cell lines and AML bone marrow mononuclear cells increased with increasing drug dose within a certain concentration range. The SLC22A3 post-intervention experiments confirmed that SLC22A3 downregulation led to active proliferation and diminished apoptosis of leukemia cells. SLC22A3 is a widely expressed drug transporter. hSLC22A3-mediated oxaliplatin uptake in cancer is thought to be important for its cytotoxicity [Our demethylation studies with DAC also showed that toxicity , but it SLC22A3 DNA is aberrant hypermethylation in AML and different clinical status of disease display distinct patterns of DNA methylation. DNA methylation levels may be useful for AML prognosis. Just as Šestáková et al. [SLC22A3 methylation level and expression in therapy of AML and to clarify whether SLC22A3 aberrant methylation facilitate or merely coexist with CEBPA mutation.In summary, this current study showed that á et al.  proposedOur results showed that increased methylation and decreased expression of SLC22A3 may be indicators of poor prognosis in AML. Methylation-silenced SLC22A3 expression may have potential guiding significance on the antileukemia effect of DAC.Additional file 1: Identification of aberrantly hypermethylated SLCs in AML. A Heatmap of differentially methylated genes between normal and AML bone marrow specimens from GSE63409. B The Venn diagram of hypermethylated genes in AML. The intersection of hypermethylated genes in AML based on GSE63409 and the RRBS data that our lab has submitted to NCBI SRA databases previously (accession number PRJNA670308). C represents normal donors.Additional file 2: Correlation between DNA methylation and mRNA expression of SLCs in AML from TCGA database. A-D, SLC5A8, SLC6A11, SLC7A14, SLC34A2. The values of zero were excluded from log calculation.Additional file 3: SLC22A3 expression in genetics subsets of Beat AML cohort. A, SLC22A3 expression in subsets of recurrent genetic abnormalities. B, SLC22A3 expression in subsets of MLL rearranged AML. C-E, SLC22A3 expression in AML with CEBPA biallelic, NPM1, and FLT3-ITD mutations.Additional file 4: Apoptotic analysis of K562 siNC/siSLC22A3 treated by DAC. A, B, Comparison of cell apoptosis between K562 siSLC22A3 and siNC after DAC dosing.Additional file 5: Differently expressed mRNAs between lower and higher SLC22A3 expression groups from beat AML.Additional file 6: Differently expressed mRNAs between lower and higher SLC22A3 expression AML groups from TCGA.Additional file 7: Differently expressed microRNAs between lower and higher SLC22A3 expression AML groups from TCGA.


IP3Rs are of particular interest because their presence provides a scaffold for ERMCs that mediate local calcium signaling, and their function outside of ERMCs depends on their motility. Unexpectedly, in a cell model with little ERMC Ca2+ coupling, IP3Rs captured at mitochondria promptly mediate Ca2+ transfer, stimulating mitochondrial oxidative metabolism. The Ca2+ transfer does not require linkage with a pore-forming protein in the outer mitochondrial membrane. Thus, motile IP3Rs can traffic in and out of ERMCs, and, when ‘parked’, mediate calcium signal propagation to the mitochondria, creating a dynamic arrangement that supports local communication.Endoplasmic reticulum-mitochondria contacts (ERMCs) are restructured in response to changes in cell state. While this restructuring has been implicated as a cause or consequence of pathology in numerous systems, the underlying molecular dynamics are poorly understood. Here, we show means to visualize the capture of motile IP The formation and dissolution of ER-Mitochondria contacts is unclear. Here, authors show that the IP3 receptor traffics in and out of the contacts and, when trapped, improves calcium signaling to stimulate energy metabolism. Moreover, the studies of ERMCs have served as a template for studies of other interorganellar junctions7.Local communication between intracellular organelles at membrane contacts is a burgeoning topic in cell biology and medicine. The contacts between the endoplasmic reticulum (ER) and mitochondria (ERMCs) have been revealed as central to a broad range of functions, including lipid biosynthesis, calcium signaling, cell survival regulation, and organelle dynamics and turnover. ERMCs have been documented in many different cell types and tissues, and compelling evidence supports their broad health relevance8. Dozens of proteins have been implicated in ERMC tethering9. The creation of synthetic linkers has allowed perturbation and measurement of contacts and local communication in live cells, and these studies have provided direct evidence for local Ca2+ and reactive oxygen species signaling at ERMCs12. It has also been shown that ERMCs tighten in response to various forms of cellular stress8 and that mobile mitochondria are retained close to the ER during cytoplasmic [Ca2+] ([Ca2+]c) elevations and might locally support the energy supply and [Ca2+]c buffering13. Thus contacts seem to dynamically restructure to meet the changing demands of the cell, but how they are formed and dissolved is yet to be determined.ERMCs are supported by protein tethers which show varying lengths and shapes2+ transport from the ER to the mitochondria that results in the mitochondrial matrix [Ca2+] ([Ca2+]m) signals to stimulate ATP production and determine cell fate. IP3Rs are IP3-and Ca2+-regulated Ca2+ release channels of the ER that mediate intracellular Ca2+ mobilization and that, together with the ensuing store-operated Ca2+ entry (SOCE), give rise to [Ca2+]c signals upon stimulation of cells by a wide range of hormones and neurotransmitters. IP3R channel function is also modulated by ATP, phosphorylation, redox, and by a large number of interacting proteins, including IRBIT, Bcl-2, and Bcl-xL. There are three distinct isoforms of IP3R, at least one of which is expressed in almost all cell types. The different IP3R isoforms show heterogeneity in tissue and intracellular distribution, structure, and regulation by IP3, Ca2+, and other factors, but have similar Ca2+ conductances and gating kinetics18.One of the most investigated inter-organelle communication mechanisms is the inositol 1,4,5-trisphosphate receptor (IP3R) mediated Ca3-induced [Ca2+]c transients are propagated to the mitochondria through the mitochondrial Ca2+ uniporter (mtCU). During IP3R-mediated Ca2+ release from the ER, a [Ca2+] nanodomain, which can be an order of magnitude higher than the bulk [Ca2+]c, is generated at the ERMCs19. This Ca2+ signal crosses the outer mitochondrial membrane (OMM), presumably through the abundant β-barrel forming OMM proteins , into the intermembrane space (IMS). Ca2+ entry into the matrix through mtCU, driven by the inside negative IMM potential (ΔΨm), is steeply dependent on [Ca2+]IMS to effectively filter out small fluctuations while responding robustly to larger [Ca2+] elevations21.IP2+ fluxes but also participate in forming close ERMCs via interaction with OMM proteins. IP3R1 and IP3R3 have been shown to co-immunoprecipitate with the OMM resident VDAC1  and the human cervical cancer cell line (HeLa). In DT40 cells, the loss of close contacts was completely rescued by the reintroduction of any single IP3R isoform, independent of its function as a Ca2+ channel25. Notably, IP3R2 showed an advantage over IP3R1 and IP3R3 in populating ERMC and mediating the ER-mitochondrial Ca2+ transfer25. However, the respective role of each IP3R isoform in the context of ERMC Ca2+ transfer has never been systematically tested in mammalian cells.Previous studies suggest that IP3Rs are not only needed for CaC1 refs. , 23, 24 C1 refs.  and TOM73/Ca2+ signaling stimuli close to the plasma membrane (PM), whereas motile IP3R were found not to release Ca2+26. To transition to the immobile and Ca2+ release competent state, the IP3R1 undergoes a ‘licensing’ procedure27. Recent evidence indicates that KRAS-induced actin-interacting protein (KRAP) determines which IP3R1s respond by tethering IP3R clusters to actin alongside sites where SOCE occurs, licensing them to evoke Ca2+ puffs and global [Ca2+]c signals. These data implicate the actin cytoskeleton in IP3R regulation28. Interestingly, actin polymerization has also been suggested to play a role in ER-mitochondrial Ca2+ transfer29, but dynamic visualization or perturbation of IP3Rs at ERMCs has been difficult to achieve.IP3Rs exhibit both immobile and motile states. Clusters of immobile IP3R1s have been shown to respond to IP2+ transfer30. After establishing the ERMC phenotype associated with the rescue of each IP3R isoform in IP3R-deficient deficient HEK cells, we utilized a synthetic linker strategy to trap the mobile IP3Rs close to the mitochondria and study IP3R dynamics in the context of [Ca2+]m and oxidative metabolism.We, here, tested each IP3R isoform’s coupling to the mitochondria in mammalian, HEK293 (HEK) cells that inherently display limited ER-mitochondrial Ca2+ handling in wild-type HEK cells (WT HEK) and in a clone lacking all three IP3R isoforms (TKO)31. Cells were transfected with a mitochondrial matrix-targeted Ca2+ sensor Cepia3 (mtCepia3) and loaded with Fura-2/AM to monitor [Ca2+]m simultaneously with [Ca2+]c. Upon addition of an IP3-generating agonist, carbachol, WT cells responded with a rapid [Ca2+]c increase which was followed by a modest increase in [Ca2+]m   quenching  Figs.  and S1D,ing Fig. .2+ transfer, we analyzed the fraction of the mitochondrial surface involved in ERMCs (0–100 nm) by transmission electron microscopy (TEM). We observed close ER-mitochondrial associations in both WT and TKO cells, however, TKO cells had fewer close contacts in the 0–20 nm range  IP3R-mediated ER Ca2+ release, (2) IP3R-dependent ER-mitochondrial contacts with the gap width relevant for Ca2+ transfer, and (3) effective mitochondrial Ca2+ sequestration. The IP3R TKO cells provide a suitable platform for testing if the ER-mitochondrial Ca2+ coupling can be manipulated by the expression of any individual IP3R isoform.To assess the structural context of the local ER to mitochondria Cange Fig. , consist3 and Ca2+31 and different in their ability to tether and communicate with mitochondria25. By re-expressing IP3Rs in the TKO cells, we studied the involvement of each individual isoform on ER-mitochondrial Ca2+ signal propagation. Acute rescue of TKO cells with each IP3R isoform completely rescued the agonist-induced [Ca2+]c responses at 48 h, but the Ca2+ signal propagation to the mitochondria, measured by mtCepia3 remained weak, similar to WT , while there was no significant change in [Ca2+]c transients in the IP3R2 and IP3R3 rescue . Specifically, we tested whether there are differences in the lateral mobility of different IP3R isoforms in the ER membrane and if the mobility is influenced by IP3-generating agonist (carbachol) stimulation. TKO cells were rescued with mCherry-tagged IP3R1 or mRFP-tagged IP3R2 or IP3R3. After rapidly photobleaching a 5 µm diameter circular area, we monitored the recovery of fluorescence in the bleached area. Although all isoforms presented a remarkable recovery of fluorescence, IP3R1 and 3 had a slightly higher mobile fraction than IP3R2  as a mask  in fixed TKO cells expressing IP3R3-HA-FRB and OMM-FKBP with and without rapamycin-induced linkage (10 min). HA-tag was labeled with AF647 and the endogenous mtHSP70 (mitochondria) with CF568. Untreated cells showed a reticular-like distribution of IP3Rs, diffusely scattered around mitochondria, in contrast to the rapamycin-treated cells where IP3R3 tightly surrounded the OMM Fig. . We alsoion Fig. . In agreion Fig. . Thus, a2+ and that, IP3Rs require “licensing” to mediate Ca2+ fluxes upon immobilization27. Thus, the question arises: Can IP3Rs immobilized at the ERMCs support local Ca2+ transfer to the mitochondria  using Fura-2 and IMS-targeted RCaMP in TKO cells expressing FRB-IP3R3 and OMM-FKBP for 48 h. These experiments were performed in the absence of extracellular Ca2+, to eliminate non-ER-derived Ca2+ responses. The drug-induced linkage between IP3R3 and the OMM at the contact sites did not alter the cytosolic response to carbachol, but significantly increased the [Ca2+]IMS , indicating that the OMM-linked IP3R3s were active upon capture at the contacts. Importantly, recruiting a generic ER membrane anchor unrelated to the IP3R (Sac1) to the OMM failed to improve the Ca2+ signal propagation to the mitochondria in WT HEK cells H autofluorescence, a readout of the activity of Ca2+-sensitive matrix dehydrogenases that are central to ATP production and the maintenance of mitochondrial redox balance. First, we analyzed the signal localization of NAD(P)H autofluorescence. We evaluated rapamycin-treated, IP3R3-linker transfected (mRFP expressing), and non-transfected cells on the same coverslip  stimulation 34 could be an unrecognized factor controlling local Ca2+ transfer to the mitochondria. Since we used either the AKAP1 transmembrane domain or TOM70 as the OMM binding partner for the IP3R, our results also support that VDAC does not have to be directly involved in the IP3R-dependent, ER-mitochondrial tether. Thus, further evaluation of the broadly accepted concept that an IP3R-GRP75-VDAC complex forms a Ca2+ tunnel between ER and mitochondria22 might be needed. Because VDACs are the most abundant protein in the OMM, it is possible that anchoring IP3R at arbitrary OMM sites might be sufficient for Ca2+ transmission. The “Ca2+ tunnel” concept is also not entirely consistent with the earlier finding that a fast Ca2+ chelator could intercept the local IP3R-mitochondrial Ca2+ transfer35. Notably, our project was made possible by the distinctive impotence of ER-mitochondrial Ca2+ coupling in HEK cells, so further studies will be needed to determine what aspects of the IP3R distribution or regulation distinguish cell types that display robust IP3R-mitochondria Ca2+ signal propagation.[Ca2+] Fig. . This re2+-sensitive matrix dehydrogenases , with 4.5 g/l glucose  supplemented with 10% FBS, 2 mM l-glutamine (Invitrogen), 100 units/ml penicillin/streptomycin , in 5% CO2 and 95% air at 37 °C.Wild-type (WT), IP3R-deficient (TKO) HEK293 and rIP3R1, mIP3R2, rIP3R3, mIP3R2GS3 stable rescue TKO HEK29336. The construction resulted in a four amino acid linker (GSRA) between the FRB and the IP3R. Our efforts to create an HA-FRB-IP3R2 construct with the strategy used for IP3R3 failed.mRFP-HA-FRB-IP3R3 were created by replacing the sequence of Cherry protein in a Cherry-rIP3R3 construct with the mRFP- and HA-tagged FRB-domain2+ measurements, TKO HEK cells were rescued with mammalian rIP3R1, mIP3R2, or mIP3R3. For mitochondrial [Ca2+] measurements, cells were transfected with matrix-targeted mtCepia3  or mtGCaMP6f  or IMS-targeted IMS-RCamP (Cox8a-RCaMP1_035). For IP3R3 co-localization linker experiments, cells were transfected with mRFP-HA-FRB-IP3R3, AKAP1(34–63)-FKBP-YFP, and/or ER-ApoK1(lumen)-CFP. For IP3R3-linker Ca2+ experiments, cells were co-transfected with FRB-HA-IP3R3 and TOM70-FKBP or AKAP1(34–63)-FKBP constructs. For FRAP experiments, TKO HEK cells were rescued with mCherry-rIP3R1, mRFP-mIP3R2, or mRFP-mIP3R3 constructs. To facilitate interorganellar linkage between the ER and the OMM, WT HEK cells were either co-transfected with mRFP-FRB-ER(Sac1)/mRFP-FRB-9x-FRB-ER(Sac1) and AKAP1(34–63)-FKBP-mRFP inducible linker pairs or AKAP1(34–63)-mRFP-(ER)Sac1/AKAP1(34–63)−9× Linker-mRFP-9x Linker-(UBC6) constitutive linkers.Cells were transiently transfected using Lipofectamine 3000 (Thermo Fisher) with 1–3 μg total DNA following the manufacturer’s protocol. For CaCells were treated with rapamycin for 10 min, followed by a washout and treatment with FK506 to prevent further co-localization. Cells were imaged shortly after this treatment.4P2O7, 2 mM Na3VO4, 1% Triton X-100 (v/v), 0.5% sodium deoxycholate (w/v), and 10% glycerol with a mixture of protease inhibitors (Roche). Lysates were incubated on ice for 30 min, then cleared by centrifugation at 16,000×g for 10 min at 4 °C, then transferred into fresh tubes. Protein concentrations were quantified with a DC protein assay kit (Bio-Rad). Protein samples were resolved on 5–7.5% SDS-PAGE gels and then transferred to nitrocellulose membranes . Rabbit polyclonal antibodies recognizing IP3R1 (CT1) (1:5000) raised against the C-terminal 19 amino acid (aa) residues of rat IP3R1 and IP3R2 (NT2) (1:200) raised against N-terminal 320–338 aa residues in mouse IP3R2 , mouse monoclonal antibody against 22–230 aa residues of human IP3R3 , Calnexin rabbit polyclonal antibody , and mouse monoclonal Flag antibody  were used for immunoblotting. Corresponding mouse and rabbit HRP-linked secondary antibodies were obtained from Cell Signaling . Proteins were detected with chemiluminescence using an HRP substrate for CCD imaging (Azure Biosystems). Elements of the mitochondrial uniporter complex were detected using anti MCU , anti MICU1 , anti MICU2 , anti EMRE , anti HSP70  and visualized by LiCor secondary antibodies.Reagents for SDS-PAGE were obtained from Bio-Rad. Cells were harvested and washed twice with ice-cold PBS then solubilized in cell lysis buffer containing 10 mM Tris-HCl, 10 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na2PO4, 20 mM Tris-HEPES at pH 7.2, and supplemented with proteases inhibitors , 2 mM MgATP, 2 μM thapsigargin (Enzo) and maintained in a stirred thermostated cuvette at 35 °C. Fura-2-FF  was added to the suspension to monitor [Ca2+] in the medium, and 1.5 μM TMRM (Invitrogen) to monitor corresponding changes in the Δψm. Assays were performed in the presence of 20 μM CGP-37157 (Enzo) and 2 mM succinate. Fluorescence was recorded in a multi-wavelength excitation dual-wavelength emission fluorimeter  using 340–380 nm excitation and 500-nm emission for fura-2-FF and 545 nm excitation and 580 nm emission for TMRM. Five data triplets were obtained per second. Ca2+-clearance was calculated after the addition of 20 μM Ca2+ pulse and complete depolarization (maximum de-quench of TMRM fluorescence) was elicited using the protonophore FCCP (2 μM). The Fura signal was calibrated at the end of each measurement, adding 1 mM CaCl2, followed by 10 mM EGTA/Tris, pH 8.5.Saponin-permeabilized HEK cells were resuspended in 1.5 ml of intracellular medium containing 120 mM KCl, 10 mM NaCl, 1 mM KH4 and stained with 0.5% uranyl acetate, pelleted in 2% agarose , dehydrated in a dilution series of acetone/ water, and embedded in Spurr’s resin (Electron Microscopy Sciences). The sections were examined with a JEOL JEM1010 TEM fitted with a side-mounted AMT XR- 50 5Mpx CCD camera or an FEI Tecnai 12 TEM fitted with a bottom-mounted AMT XR-111 10.5 Mpx CCD camera. Whole-cell images were taken with ×6700–14,000 (JEOL) or ×4400–6500 (FEI) direct magnification. Mitochondria were imaged using ×40,000  or ×15,000  magnification. Interfaces between ER and mitochondria were analyzed using a custom ImageJ script: https://sites.imagej.net/MitoCare/.HEK cells were fixed using 2% glutaraldehyde, then with 1% OsOFor immuno-EM experiments, TKO HEK cells were sorted by flow cytometry (Aria) for double positives of mRFP-HA-FRB-IP3R3 and OMM-FKBP-YFP. Sham-transfected control TKO cells were treated the same way as the transfected cells. An equal count of sorted cells was plated on 14 mm plastic coverslips (Thermanox) for 6 h before treatment and processing. Cells were fixed with 3% paraformaldehyde followed by quenching, permeabilization, blocking, and labeling with primary antibody , followed by secondary blocking and incubating with secondary antibody anti-rabbit IgG Nanogold . Samples were dehydrated in a graded series of alcohol and embedded in Durcupan (Electron Microscopy Sciences). After polymerization, ultrathin sections (65–80 nm) were prepared using ultramicrotome  and studied with FEI Tecnai 12 electron microscope. Images were taken at ×3200–15,000 magnification.3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, and 10 mM glucose, pH 7.4) containing 2% BSA (Roche) in the presence of 0.003% Pluronic F-127 and 150 μM sulfinpyrazone for 15 min at 37 °C to measure changes in the [Ca2+]c. After dye-loading, cells were washed with fresh ECM containing 0.25% BSA and transferred to the thermostated stage (37 °C) of the microscope. For the epifluorescence Ca2+ imaging, the ImagEM EM-CCD camera (Hamamatsu) was fitted to an Olympus IX81 microscope with Lambda DG4 light source operated by Metamorph (Molecular Devices). The imaging setups used the UV-optimized Olympus UAPO/340 ×40/1.35 N.A. oil immersion objective. For simultaneous measurements of [Ca2+]c and [Ca2+]m or [Ca2+]IMS, Fura-2 fluorescence was recorded at 340 and 380 nm, the mitochondrial matrix-targeted Cepia3 or 485/15 nm or IMS-targeted RCaMP at 577/20 nm excitations, using dual-band dichroic and emission filters . Images were acquired every 0.2 s or 0.33 s. Fura-2 was calibrated in vitro by adding 1 mM CaCl2, followed by 10 mM EGTA/Tris, pH 8.5. [Ca2+]c was determined by the following formula: [Ca2+] = Kd × (Sf2/Sb2) × (R − Rmin)/(Rmax − R), where Kd is the Ca2+-dissociation constant (0.224 μM), R is the ratio of the fluorescence intensities at 340/380 nm excitation, Rmin and Rmax are the fluorescence ratios in Ca2+-free and Ca2+-saturated conditions, respectively, Sf2/Sb2 is the ratio of fluorescence intensities measured at 380 nm excitation in Ca2+-free (f)/Ca2+-saturated  conditions.HEK and HeLa cells plated on Poly-D-Lysine coated round (25 mm) coverslips (Fisher Cat#) were loaded with 2 μM fura-2/AM (Invitrogen) in a serum-free extracellular medium  was used to measure NAD(P)H autofluorescence. A high-pressure Xenon lamp was used for illumination with 350/20 nm excitation, 400 nm long-pass dichroic and 460/50 nm emission filters. Hardware synchronization and image acquisition was controlled through a Windows PC using custom software .Zeiss LSM 880 confocal system equipped with Airyscan detector, using 488, 561, and 633-nm laser lines for green, red, and far-red fluorescent probes, respectively. Images were taken through a ×63 oil objective . Images were captured using Carl Zeiss ZEN 2.3 software package (Carl Zeiss Microscopy GmbH 1997–2015) and were processed using ImageJ .FRAP experiments were performed on a Zeiss LSM780 NLO system using a 63x oil immersion objective . DPSS laser at 561 nm was used for the excitation of mRFP/mCherry. Circular regions 5 µm in diameter were photobleached by pulsed Ti:Sapphire laser (Coherent Chameleon Vision II) at 720 nm to achieve rapid, extensive bleaching. Images were acquired at 1 frame/s using Carl Zeiss ZEN 2.3 software package (Carl Zeiss Microscopy GmbH 1997–2015) and were processed using ImageJ .3D super-resolution microscopy was performed using a Vutara 352 nanoscope (Bruker). Briefly, two focal planes were projected onto the sensor of an ORCA Flash v.2 sCMOS camera (Hamamatsu) through an Olympus UPlanSApo ×60/1.2 N.A. water-immersion objective. The point spread functions for the wavelengths used were calibrated and aligned using 100 nm Tetraspeck microspheres (Thermo Fisher Scientific). Laser illumination at 640 and 561 nm were used for stochastic imaging of AlexaFluor 647 and CF568 (Biotium), respectively, and 405 nm light was used to recover portions of CF568 from their dark state. The two colors were recorded sequentially  and several thousand frames were acquired for each fluorophore. Cells were plated, fixed and IP3R-HA were labeled with HA  primary then AF647  and mtHSP70 (JG1)  primary then with CF568 Goat Anti-Rabbit IgG  secondary antibodies. Imaging was performed in freshly prepared buffer for oxygen- depleting and reducing conditions: 20 mM cysteamine, 1% 2-mercaptoethanol (v/v), 169 AU/ml glucose oxidase and 1400 AU/ml catalase in 50 mM Tris-HCl, 10 mM NaCl, and 10% (w/v) glucose (pH 8.0). Calibration, alignment, acquisition, and localization were all done in the Vutara SRX software.t-test with two-tailed p values. If not, Mann–Whitney rank sum test (two groups) or Kruskal–Wallis one-way ANOVA on ranks (three or more groups) with post hoc Dunn’s test was used. p < 0.05 was considered significant for all tests. All graphs were created in SigmaPlot 12 or Prism 9 and were combined with the images in Microsoft PowerPoint and Adobe Illustrator. Illustrations were created with Adobe Illustrator and BioRender (BioRender.com).Statistical testing was performed using either SigmaPlot , Prism , or Excel (Microsoft). For comparisons between groups, first, it was determined whether the data were normally distributed using the Shapiro–Wilk test (SigmaPlot). If data were normally distributed, one-way ANOVA was used with post hoc Holm–Sidak test for pairwise comparisons or an unpaired Every element of each graph and image was created by the Authors. No previously created elements were used.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


P=0.003). Moreover, ACOT2 exhibited excellent diagnostic efficiency for AML (AUC: 1.000) and related to French-American-British (FAB) classification and cytogenetics. GO, KEGG, and GSEA analyses of 71 common targets between ACOT2 coexpressed and AML-related genes revealed that ACOT2 is closely related to ACOT1, ACOT4, enoyl-acyl carrier protein reductase, mitochondrial (MECR), puromycin-sensitive aminopeptidase (NPEPPS), SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), and long-chain fatty acid-CoA ligase 1 (ACSL1) in PPI network, and plays a significant role in lipid metabolism, that is, involved in fatty acid elongation and biosynthesis of unsaturated fatty acids. Collectively, the increase of ACOT2 may be an important characteristic of worse OS and abnormal lipid metabolism, suggesting that ACOT2 may become a potential therapeutic target for AML.Acyl-CoA thioesterase (ACOT) plays a considerable role in lipid metabolism, which is closely related to the occurrence and development of cancer, nevertheless, its role has not been fully elucidated in acute myeloid leukemia (AML). To explore the role of ACOT2 in AML and to provide a potential therapeutic target for AML, the expression pattern of ACOT was investigated based on the TNMplot, Gene Expression Profiling Interactive Analysis (GEPIA), and Cancer Cell Line Encyclopedia (CCLE) database, and diagnostic value, prognostic value, and clinical phenotype of ACOT were explored based on data from The Cancer Genome Atlas (TCGA). Functional annotation and enrichment analysis of the common targets between ACOT2 coexpressed and AML-related genes were further performed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) analyses. The protein-protein interaction (PPI) network of ACOT2 coexpressed genes and functional ACOT2-related metabolites association network were constructed based on GeneMANIA and Human Metabolome Database. Among ACOTs, ACOT2 was highly expressed in AML compared to normal control subjects according to TNMplot, GEPIA, and CCLE database, which was significantly associated with poor overall survival (OS) in AML ( Acute myeloid leukemia (AML) is a neoplastic disease originating from myeloid progenitor cells characterized by malignant proliferation and differentiation, which is often accompanied by proliferative diseases such as severe leukocytosis –3. AlthoHunt et al. , 10 idenhttps://tnmplot.com/) [The expression level of the type I ACOT family including ACOT1, ACOT2, ACOT4, and ACOT6 were evaluated using the TNMplot platform (ot.com/) , which ihttps://gepia.cancer-pku.cn/) [Gene expression profiling interactive analysis   is a newhttps://www.broadinstitute.org/ccle) [The expression of ACOT1, ACOT2, ACOT4, and ACOT6 originated from type I ACOT family in AML cell lines was verified by the Cancer Cell Line Encyclopedia  , which grg/ccle) . The CCLP < 0.05). The receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic value. The area under the ROC curve (AUC) was used to summarize diagnostic accuracy, with 1.0 representing perfect discrimination and 0.5 representing chance discrimination.The diagnostic and prognostic values of ACOT1, ACOT2, ACOT4, and ACOT6 expression were evaluated in patients with AML based on bone marrow RNA-seq dataset data acquired from TCGA and GTEx databases. The samples were divided into high and low expression groups according to the median expression, and the effects of high and low expression on AML prognosis were analyzed. The overall survival (OS) curve was drawn by a Kaplan–Meier method. The “survival” package (version 3.2-10) was applied for statistical analysis of survival data, and “survminer” package (version 0.4.9) was used for visualization. The log-rank test was applied to evaluate the statistical significance of differences in survival duration  blasts, peripheral blood (PB) blasts, white blood cell (WBC) counts, cytogenetic risks, French-American-British (FAB) classifications, cytogenetics, FLT3 mutation, IDH1 R132 mutation, IDH1 R140 mutation, IDH1 R172 mutation, RAS mutation, and NPM1 mutation was analyzed based on logistic regression models. This analysis was conducted by taking ACOT2 as independent variables, and relevant clinical characteristics were set as dependent variables.https://www.linkedomics.orglogin.php) [https://ctdbase.org/) [https://bioinfogp.cnb.csic.es/tools/venny/index.html).LinkedOmics (gin.php)  is a newse.org/)  was usedhttps://biit.cs.ut.ee/gprofiler/gost) [P < 0.05 was considered statistically significant for GO annotation enrichment analysis and KEGG pathway enrichment analysis.The GO resource provides a platform for functional annotation and enrichment analysis of genes. KEGG is a comprehensive database of biological information designed to assist in the interpretation of large-scale molecular datasets. In the present study, the top 30 core targets of functional protein association network were screened, and GO biological function enrichment analysis and KEGG metabolic process analysis were performed based on g:Profiler (er/gost) . P < 0.0P < 0.05 was considered to be significantly enriched. The Hallmark list from the h.all.v7.2.symbols.GMT was used as the gene set reference.In order to further understand the functional implications of ACOT2, the gene expression files of the 151 cases downloaded from TCGA were separated into high-express groups and low-express groups based on “DESeq2” package (version 1.26.0). In accordance with the differential analysis of high and low expression groups, we sorted the differentially expressed genes (DEG) according to log fold change, then constructed the ordered gene set and enriched it with GSEA based on “clusterProfiler” package (version 3.14.3). https://genemania.org) [GeneMANIA (nia.org)  is a flehttps://hmdb.ca/) [https://www.cytoscape.org) (version 3.8.1). ACOT2-related metabolites were input into Cytoscape software to construct a functional ACOT2-related metabolites association network.The Human Metabolome Database (mdb.ca/)  was usedP < 0.05). The approach directly compared AML and normal samples by grouping all specimens of the same category, and the results were visualized by violin plots. The normal AML comparison was shown in Figures The elevated expression of the type I ACOT  in AML subjects was observed in the TNMplot database  (P=0.091) (P=0.193) (P=0.567)  in AML. One hundred and fifty-one AML samples and corresponding RNA-seq dataset were obtained from TCGA database, however, the information on survival time for 11 samples was missing. Consequently, 140 samples were included in the analysis. The cutoff value of median ACOT1, ACOT2, ACOT4, and ACOT6 expressions were 0.885577317, 2.972574801, 1.491169926, and 0.558179678, respectively. Patients were split into high- and low-expression groups. Results indicated that high expression of ACOT2 was correlated with a significantly poorer prognosis compared with those exhibiting low ACOT2 expression (hazard ratio (HR) = 1.90; P=0.003) , whereasP=0.091) , ACOT4 (P=0.193) , and ACOP=0.567)  was not P=0.567) , ACOT2 (P=0.567) , and ACOP=0.567)  exhibiteP=0.567)  on AML wThe correlation between ACOT2 expression levels and clinical characteristics, including age, gender, patients' race, WBC counts, BM blasts, PB blasts, cytogenetic risks, and FAB classification were analyzed, and the results suggested that ACOT2 did not differ significantly with most of these clinical characteristics in AML except for FAB classification and cytogenetics . CompareNext, the function module of LinkedOmics database was applied to explore the coexpressed genes of ACOT2 in AML. The top 50 significant genes either positively or negatively correlated with ACOT2 were screened out in the heatmap Figures . As showP=0.046) (P=0.046) (P=0.039) (P=0.046)  biological process enrichment analysis of 71 common targets between ACOT2 coexpressed and AML-related genes indicated that the processes were involved in the catalytic activity, myristoyl-CoA hydrolase activity, carboxylic ester hydrolase activity, palmitoyl-CoA hydrolase activity, acyl-CoA hydrolase activity, and CoA hydrolase activity at the MF levels, cytoplasm at the CC levels, and very long-chain fatty acid metabolic process, organonitrogen compound metabolic process, cellular amide metabolic process at the BP levels in AML . The KEGP=0.046) , heme meP=0.046) , interfeP=0.039) , myogeneP=0.046) . These fThe 71 common targets between ACOT2 coexpressed and AML-related genes were uploaded to the GeneMANIA for the protein-protein interactions of common genes analysis . Among tIn the present study, we obtained 53 ACOT2-related metabolites based on Human Metabolome Database . Our further research showed that the expression of ACOT2 was significantly upregulated in most 26 AML cell lines, especially in HNT-34, MOLM-16, BDCM, and THP-1 cell lines. However, there was no significant increase in ACOT1 and ACOT4 in almost any of the AML cell lines, and the expression of ACOT6 in 26 AML cell lines was not available. Survival analysis indicated that the increased level of ACOT2 (P=0.003) was significantly associated with poor OS in AML, whereas ACOT1 (P=0.091), ACOT4 (P=0.193), and ACOT6 (P=0.567) were not significantly correlated. Moreover, the receiver conducting feature curve analysis indicated that ACOT2 (AUC: 1.000) was a prediction of AML and exhibited excellent diagnostic efficiency for AML. Hence, highly expressed ACOT2 is a valuable prognostic factor for AML, indicating that inhibiting the expression of ACOT2 may be a potential approach to prevent the development of AML.So far, metabolic abnormalities have been observed in many tumor diseases, including AML. Nevertheless, there are few cases of specific metabolic enzyme-related gene changes that have been investigated, and the expression and clinical significance of ACOT2 in AML have not been revealed. Here, our research reported that the upregulated expression of ACOT2 and abnormal lipid metabolism were closely related to the clinical prognosis of AML, suggesting that ACOT2-dependent abnormal lipid metabolism may become a prospective target to evaluate the clinical significance of AML diseases. According to the data of TNMplot and GEPIA database, type I ACOT  was highly expressed in AML (all Then, we further studied the correlation between ACOT2 expression and clinical characteristics of AML, and the results suggested that ACOT2 did not differ significantly with most of these clinical characteristics in AML, except for FAB classification and cytogenetics. Compared to the favorable cytogenetic risk group, ACOT2 was highly expressed in intermediate and poor cytogenetic risk groups. Additionally, we examined the association between ACOT2 expression and mutated genes in AML with FLT3, IDH1 R132, IDH1 R140, IDH1 R172, RAS, and NPM1, but no significant correlations were found.Next, the function module of LinkedOmics database was applied to explore the coexpressed genes of ACOT2 in AML, and the top 50 significant genes positively or negatively correlated with ACOT2 were screened out. GO, KEGG, and GSEA analysis of 71 common targets between ACOT2 coexpressed and AML-related genes revealed that the involved processes were the catalytic activity, palmitoyl-CoA hydrolase activity, acyl-CoA hydrolase activity, and CoA hydrolase activity, and very long-chain fatty acid metabolic process, organonitrogen compound metabolic process, cellular amide metabolic process, the most enriched pathways included fatty acid elongation and biosynthesis of unsaturated fatty acids, and the gene sets were significantly enriched in estrogen response early, heme metabolism, interferon alpha response, myogenesis. The PPI network results showed that ACOT2, ACOT1, ACOT4, MECR, NPEPPS, SMARCB1, and ACSL1 played a considerable role in the PPI network, which is related to lipid metabolism. Besides, the ACOT2 and its metabolites interaction network indicated that these metabolites are closely related to lipid metabolism, suggesting that ACOT2 plays significant roles in lipid metabolism involved in fatty acid elongation and biosynthesis of unsaturated fatty acids. Although the metabolites interaction network has greatly expanded the interaction relationships of multiple metabolites, there are still many members whose functions are relatively unexplored and deserve further study.In this study, we explored the mechanism by which high expression of ACOT2 predicts decreased overall survival and abnormal lipid metabolism during AML disease progression. The expression of ACOT2 was significantly upregulated in AML cell lines, which had good diagnostic performance and was significantly associated with poor OS in AML. Besides, ACOT2 plays a significant role in lipid metabolism such as fatty acid elongation and biosynthesis of unsaturated fatty acids. In conclusion, the high expression of ACOT2 may be an important characteristic of decreased overall survival and abnormal lipid metabolism in AML, and the increase of ACOT2 in AML can be used as a potential biomarker for auxiliary diagnosis and prognosis, and targeted ACOT2 may be a promising approach to inhibit AML progression.


Coordinated endothelial control of cardiovascular function is proposed to occur by endothelial cell communication via gap junctions and connexins. To study intercellular communication, the pharmacological agents carbenoxolone (CBX) and 18β‐glycyrrhetinic acid (18βGA) are used widely as connexin inhibitors and gap junction blockers.2+ waves, evoked by inositol 1,4,5‐trisphosphate (IP3) in the endothelium of intact mesenteric resistance arteries.We investigated the effects of CBX and 18βGA on intercellular Ca3‐mediated Ca2+ release and propagated waves were inhibited by CBX (100 μM) and 18βGA (40 μM). Unexpectedly, the Ca2+ signals were inhibited uniformly in all cells, suggesting that CBX and 18βGA reduced Ca2+ release. Localised photolysis of caged IP3 (cIP3) was used to provide precise spatiotemporal control of site of cell activation. Local cIP3 photolysis generated reproducible Ca2+ increases and Ca2+ waves that propagated across cells distant to the photolysis site. CBX and 18βGA each blocked Ca2+ waves in a time‐dependent manner by inhibiting the initiating IP3‐evoked Ca2+ release event rather than block of gap junctions. This effect was reversed on drug washout and was unaffected by small or intermediate K+‐channel blockers. Furthermore, CBX and 18βGA each rapidly and reversibly collapsed the mitochondrial membrane potential.Acetycholine‐evoked IP3‐mediated Ca2+ release and depolarise the mitochondrial membrane potential. These results suggest that CBX and 18βGA may block cell–cell communication by acting at sites that are unrelated to gap junctions.CBX and 18βGA inhibit IP Changes in 3IP and cytoplasmic Ca2+ concentration decode information held in extracellular activators and encode intracellular signals that regulate the production of NO, prostacyclin and signalling peptides that diffuse to smooth muscle cells  in facilitating cell–cell communication and the transmission of Ca18β‐glycyrrhetinic acid (18βGA) and its derivative carbenoxolone (CBX). Derived from the liquorice root Glycyrrhiza glabra, 18βGA . cIP3 provides precise spatial and temporal control of the site of cell activation and Ca2+ release. Paired cellular responses to ACh or cIP3 were analysed before and after various pharmacological interventions with CBX and 18βGA. Intercellular Ca2+ waves were blocked by CBX and 18βGA, but this occurred by inhibition of IP3‐evoked Ca2+ release rather than block of gap junction‐mediated signal propagation. The inhibition of IP3‐evoked Ca2+ release by CBX and 18βGA was reversible and was unaffected by the presence of small or intermediate K+‐channel blockers. Furthermore, CBX and 18βGA each also rapidly and reversibly collapsed the mitochondrial membrane potential. These results suggest that CBX and 18βGA act at sites outwith gap junctions by inhibiting IP3‐mediated Ca2+ release and depolarising mitochondrial membrane potential (ΔΨM). Care is required in the use of these drugs when IP3‐mediated Ca2+ signalling is being investigated.To investigate whether or not gap junctions play a role in endothelial IP22.1British Journal of Pharmacology  Act 1986, UK]. Animal studies are reported in compliance with the ARRIVE guidelines , from an in‐house colony, were used for the study. The animals were housed three per cage, and the cage type was North Kent Plastic model RC2F with nesting material “Sizzle Nest.” A 12:12 light dark cycle was used with a temperature range of 19–23°C (set point 21°C) and humidity levels between 45% and 65%. Animals had free access to fresh water and SDS diet RM1 (rodent maintenance). The enrichment in the cages was aspen wood chew sticks and hanging huts.Animals were killed by cervical dislocation and the mesenteric bed removed. All experiments were performed using first‐ or second‐order mesenteric arteries. Controls and experimental treatments were carried out in the same tissue, so blinding and randomisation were not used.2.2+ PSS (composition below) was used in all experiments. Endothelial cells were loaded with the Ca2+ indicator dye Cal‐520  and then mounted in a custom flow chamber . This system was used for K+‐channel blocking experiments. All images were acquired by Andor iXon EMCCD cameras (1024 × 1024) using MicroManager v1.4.22  and custom designed, dual FITC/TRITC filter sets Figure . A 40× 12.42+ responses were evoked by photolysis of caged IP3 the endothelium was dual loaded with Cal‐520/AM and with a membrane‐permeant caged IP3  for 30 min at 37°C  at 300 V, which produced light of ~1 ms duration. The flashlamp output was passed through a 395 nm short pass filter into a 1250 μm diameter light guide , 0.2 , or 0.1 . These experiments were performed such that the 27% transmission was recorded first, followed by 63% transmission with 15 min rest between photolysis events, and so on.In some experiments, the extent of IP2.53‐mediated Ca2+ release, ACh‐ or cIP3‐evoked endothelial Ca2+ activity was measured at 10 Hz. Baseline Ca2+ activity was recorded for 30 s, and then endothelial Ca2+ activity evoked by ACh (50 nM) or photolysis of cIP3. The same arteries were then incubated with CBX  or 18βGA . ACh/cIP3‐evoked Ca2+ activity was then recorded again. In separate experiments, this protocol was repeated with an additional washout period of 1 h  before an additional recording was taken.In experiments that examined the effect of CBX and 18βGA on IP+‐channel blockade on endothelial Ca2+ signalling, ACh‐evoked (50 nM) Ca2+ activity was assessed in the absence and then the presence of either the KCa2.x channel blocker, apamin  or the KCa3.1 channel blocker, TRAM‐34 . After their introduction, K+‐channel blockers remained in the PSS until washout, as indicated. In all experiments, there was a minimum of 15 min between successive stimulations for responses to recover.In experiments assessing the effect of KM) was assessed using the membrane potential‐sensitive fluorophore, tetramethylrhodamine ethyl ester   was used to prevent plasma membrane potential changes.In a separate series of experiments, the effects of CBX and 18βGA on ΔΨIn experiments where cell viability was assessed, propidium iodide (1.5 μM) was added into the PSS, 100 images were acquired and an average image intensity projection generated using Fiji  was applied in a Ca2+‐free bath solution. By inhibiting SERCA, CPA disrupts the store uptake‐leak equilibrium so that the leak may be measured as a rise in cytoplasmic Ca2+ concentration and integrated to determine the store content. In these experiments, CBX was used to inhibit IP3 receptor activity and the effectiveness of block confirmed by the absence of a response to ACh (50 nM). The bathing media was then changed to Ca2+‐free PSS containing CPA and the whole‐field Ca2+ signal profile measured over the next 15 min. The area under the Ca2+ discharge curve was calculated as a measure of store Ca2+ content and compared to controls.In experiments in which Ca2.62+ signals were extracted from Ca2+ imaging data as previously described  signals were converted to baseline‐corrected fluorescence intensity (F/F0) by dividing each intensity measurement by the average value of a 100‐frame baseline period at the start of each trace. F/F0 signals were smoothed using a 21‐point third‐order polynomial Savitzky–Golay filter, and key signal parameters  extracted automatically. Analysis of cIP3‐evoked Ca2+ activity was restricted to those cells in which cIP3 was photolysed. This was achieved by applying a mask restricted to the photolysis region. The photolysis region occupied a fraction of the overall field, so these experiments had a lower number of cells per experiment than those of ACh‐evoked signalling.Single‐cell Ca2+ wave propagation, we created images of active Ca2+ wavefronts by calculating ΔF/F0 for each image in the recording. For cIP3‐evoked Ca2+ experiments, a maximum intensity projection of the first 3 images immediately following uncaging was taken, ensuring that only signal from the uncaging area is presented. This only differs in Figure To visualise Ca2.7F‐test) before statistical tests were performed. Raw peak F/F0 responses were analysed statistically using either a paired Student's t‐test or a paired one‐way ANOVA with Tukey's multiple comparisons test on Prism where an appropriate F value was achieved, version 6.0 . P < 0.05 was considered statistically significant. The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology . Cal‐520/AM and TMRE were obtained from Abcam . Pluronic F‐127 was obtained from Invitrogen . TRAM‐34 and apamin were obtained from Tocris . CBX and 18βGA, ionomycin, ACh, propidium iodide and all other chemicals were obtained from Sigma . All solutions were freshly prepared each day.Caged IP2.9http://www.guidetopharmacology.org) and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 , evoked heterogeneous increases in Cas Figure . These Cs Figure . There w2+ or IP3 through gap junctions between endothelial cells  and 18βGA (40 μM) each significantly reduced the number of cells responding to ACh and the amplitude of ACh‐evoked responses Figure .2+ waves. However, the decrease in amplitude of ACh‐evoked Ca2+ signals occurred approximately uniformly across all endothelial cells—an unexpected finding, as these drugs would not be expected to reduce Ca2+ signals in cells directly activated by ACh. These results raised the possibility that CBX and 18βGA may each directly inhibit IP3‐evoked Ca2+ release.These results initially appeared to be consistent with a contribution of gap junctions to the propagation of endothelial cell Ca3 to evoke Ca2+ release, the effects of the drugs on Ca2+ signals evoked by the photolysis of cIP3 were examined . This experimental protocol was not performed for CBX as the drug's inhibition of Ca2+ signalling was too rapid (<10 min). 18βGA evoked a time‐dependent reduction in (1) the amplitude of cIP3‐evoked Ca2+ signals within the photolysis site and (2) the outward propagation of Ca2+ signals from the photolysis site , there was a reduction in the amplitude of the Ca2+ response in the photolysis site and in the subsequent outward propagation of Ca2+ waves  of CBX  and intermediate (IK) conductance K2+ signals, while CBX abolished the response in these same preparations  as measured using the area under the whole field Ca2+ signal intensity curve upon addition of CPA  in a Ca2+‐free PSS   and reversible depolarisation of ΔΨM  when used at concentrations and incubation times reported to block gap junctions  or CBX (up to 100 μM)  inhibited ACh‐mediated Ca2+ release in the intact mouse aortic endothelium  blocked Ca+‐channel activity  or IK‐channel blocker (TRAM 34) on IP3‐mediated Ca2+ release. This suggests that inhibition of K+‐channel activity is an unlikely explanation of CBX‐ and 18βGA‐mediated inhibition of IP3‐mediated Ca2+ release in mesenteric artery endothelium.An alteration in KM collapse induced by each of the gap junction blockers. The collapse of ΔΨM will have wide ranging effects on cell signalling. The CBX‐ and 18βGA‐induced collapse of ΔΨM (measured with TMRE) occurred in normal PSS and in a high K+ PSS that was used to clamp the plasma membrane potential. The concentration of TMRE in mitochondria is a Nernstian function of the ΔΨM and plasma membrane potential. Our finding that the change in TMRE fluorescence persisted in a high K+ PSS confirmed that CBX and 18βGA alter ΔΨM, not the endothelial plasma membrane potential.Another unexpected finding in the present study was the rapid ΔΨM. For example, in a pituitary adenoma cell line, 18βGA (up to 150 μM) caused a decrease in ΔΨM and elevated intracellular ROS and Ca2+ concentrations, stimulating mitochondrial permeability transition (MMP) leading to increased apoptosis  .While CBX and 18βGA each depolarised ΔΨg Figure  and the M by CBX and 18βGA could explain the changes in IP3‐evoked Ca2+ release  while inhibition of IP3‐mediated Ca2+ release developed more slowly . The differences in time course suggests that ΔΨM depolarisation alone does not explain the inhibition of IP3‐evoked Ca2+ release and that CBX or 18βGA block IP3 receptors.Collapse of ΔΨ3‐mediated signal transduction via gap junctions in intact arterial tissue. CBX and 18βGA each inhibit IP3‐mediated Ca2+ release and depolarise ΔΨM.Together, our study questions the usefulness of CBX and 18βGA in studies on IPC.B., C.W., and J.G.M. developed the concept. C.B. and X.Z. performed the experiments. C.B., X.Z., and C.W. analysed the data. C.B., X.Z., C.W., and J.G.M. interpreted the data. C.B. and J.G.M. drafted the manuscript. C.B., C.W., X.Z., and J.G.M. edited the manuscript. C.W., C.B., and J.G.M. sourced funding. All authors approved the final version of the manuscript.The authors declare no conflict of interest.Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting researchThis Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Figure S1:Incubation with 18βGA and CBX does not increase membrane permeabilityn face mesenteric artery preparations were stained with Cal‐520  and propidium iodide  and control recordings, recordings after incubation with (A) 18βGA  or (C) CBX , and after 1 hr washout .  The number of propidium iodide‐positive cells for each condition, plotted as a percentage of the total number of cells in the field of view. Examples from single replicates are shown from n = 5 paired biological replicates. Scale bars = 50 μm. EClick here for additional data file.Video S1: ACh evokes reproducible endothelial Ca2+increasesn face mesenteric artery preparation stained with Cal‐520  was stimulated with ACh (50 nM) after 30s baseline recording (10 Hz). The resulting Ca2+ activity is visualised in green. Upper image: repeat 1; lower image: repeat 2, taken 30 mins after repeat 1. Scale bars = 50 μm.EClick here for additional data file.Video S2: cIP3evokes reproducible endothelial Ca2+increasesn face mesenteric artery preparation stained with Cal‐520  and cIP3 (5 μM). Ca2+ activity was stimulated by localised photolysis (yellow circle) after 30s baseline recording (10 Hz). The resulting Ca2+ activity is visualised in green. Upper image: repeat 1; lower image: repeat 2, taken 30 mins after repeat 1 in the same preparation. Scale bars = 50 μm.EClick here for additional data file.Video S3: CBX inhibits cIP3‐evoked endothelial Ca2+responsesn face mesenteric artery preparation stained with Cal‐520  and cIP3 (5 μM). Ca2+ activity was stimulated by localised photolysis (yellow circle) after 30s baseline recording (10 Hz). The resulting Ca2+ activity is visualised in green. A control recording was taken (upper image) after which the preparation was left to rest for 15 mins, then incubated in CBX (100 μM) for 5 mins. A second recording was then taken (lower panel). Scale bars = 50 μm.EClick here for additional data file.Video S4: 18βGA inhibits cIP3‐evoked endothelial Ca2+responsesn face mesenteric artery preparation stained with Cal‐520  and cIP3 (5 μM). Ca2+ activity was stimulated by localised photolysis (yellow circle) after 30s baseline recording (10 Hz). The resulting Ca2+ activity is visualised in green. A control recording was taken (upper image) after which the preparation was left to rest for 15 mins, then incubated in 18βGA (40 μM) for 45 mins. A second recording was then taken (lower panel). Scale bars = 50 μm.EClick here for additional data file.Video S5: CBX rapidly depolarises the mitochondrial membrane potentialn face mesenteric artery preparation were stained with TMRE  to visualise the mitochondrial membrane potential (ΔΨM). Mitochondria were imaged for 1.5 mins whilst administering PSS only (upper panel) or PSS + CBX (100 μM) after a baseline recording of 30s at 1.5 ml.min−1 under constant flow. Scale bars = 25 μm.EClick here for additional data file.


Chlamydia trachomatis, replicates within a parasitophorous vacuole termed an inclusion. During development, host proteins critical for regulating intracellular calcium (Ca2+) homeostasis interact with the inclusion membrane. The inclusion membrane protein, MrcA, interacts with the inositol-trisphosphate receptor (IP3R), an ER cationic channel that conducts Ca2+. Stromal interaction molecule 1 (STIM1), an ER transmembrane protein important for regulating store-operated Ca2+ entry (SOCE), localizes to the inclusion membrane via an uncharacterized interaction. We therefore examined Ca2+ mobilization in C. trachomatis infected cells. Utilizing a variety of Ca2+ indicators to assess changes in cytosolic Ca2+ concentration, we demonstrate that C. trachomatis impairs host cell SOCE. Ca2+ regulates many cellular signaling pathways. We find that the SOCE-dependent NFAT/calcineurin signaling pathway is impaired in C. trachomatis infected HeLa cells and likely has major implications on host cell physiology as it relates to C. trachomatis pathogenesis.The obligate intracellular bacterium,  Chlamydiae contains the human pathogens Chlamydia trachomatis, C. psittaci, and C. pneumoniae. Chlamydiae are obligate intracellular, gram negative bacteria that undergo a biphasic developmental cycle with the bacteria in either an infectious and metabolically constrained state, termed the elementary body (EB), or a metabolically active and replicative state that is non-invasive, and termed a reticulate body (RB)2. The intracellular development of chlamydiae occurs within a parasitophorous vacuole that is referred to as an inclusion3. C. trachomatis contains multiple serological variants that demonstrate specific organotropism and associated disease. Serovars A–C are associated with endemic, blinding trachoma, serovars D–K are responsible for the common sexually transmitted urogenital infections, and serovars L1–L3 are the causative agents of lymphogranuloma venereum, a more invasive disease6.The phylum C. trachomatis utilizes a type III secretion system to deliver effectors across the inclusion membrane and into the cytosol of the host cell7. A subset of these T3SS effector proteins, termed Incs, localize to the inclusion membrane via a bi-lobed transmembrane domain9. Incs are oriented in the inclusion membrane in a manner that exposes them to the cytosol of the host cell11, enabling the Incs to interact with cytosol-exposed proteins12. While Incs are typically distributed relatively evenly around the inclusion membrane, a subset of Incs are localized to discrete, punctate sites on the membrane, termed microdomains, that are also enriched in cholesterol and active host Src-family kinases13. Currently, the C. trachomatis effectors CT101 (MrcA), CT147, CT222, CT223 (IPAM), CT224, CT228, CT232 (IncB), CT233 (IncC), CT288, and CT850 have been identified as Incs enriched in inclusion microdomains15. Early findings demonstrated microdomains function as hubs for interactions with the cytoskeleton and promote the positioning of the inclusion at the microtubule organizing center16. Microdomains also influence extrusion-based dissemination, a process in which all or part of an intact chlamydial inclusion is exocytosed from the host cell for dissemination to distal anatomic sites. The phosphorylation of myosin light chain 2 (MLC2) at the microdomain is a critical regulator of this dissemination process14, which occurs via ionized calcium (Ca2+)-dependent pathways17.2+ acts as a second messenger for a number of pathways in eukaryotes influencing fertilization, embryonic axis formation, cell differentiation, cell proliferation, transcription factor activation, and cell fate decisions19. Cytosolic Ca2+ is tightly regulated to ensure these pathways are activated only when required. This homeostatic regulation of cytosolic Ca2+ concentration is achieved through Ca2+ pumps, exchangers, channels, buffering proteins, and sensors to control Ca2+ movement across the plasma membrane and the membranes of intracellular Ca2+ stores20. In excitable and non-excitable cells, store-operated Ca2+ entry (SOCE) is a major regulator of Ca2+ homeostasis21. The SOCE process is initiated by the depletion of endoplasmic reticulum (ER) Ca2+. The reduction in ER luminal Ca2+ is sensed by stromal interaction molecule 1 (STIM1) and subsequently initiates a STIM1 conformational change enabling STIM1 to interact with Orai1 to generate Ca2+ release-activated Ca2+ (CRAC) influx channels at the plasma membrane. The STIM1-CRAC channel interaction opens the channel resulting in an ingress of extracellular Ca2+ into the cytosol22. This influx of extracellular Ca2+ into the cytosol refills Ca2+ stores via the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)23 and is a signal for select Ca2+-dependent pathways.Cytosolic CaC. trachomatis inclusion interacts with the host ER, a major intracellular Ca2+ store, through multiple interactions including the inclusion membrane protein IncD interaction with ceramide transfer protein (CERT)24, the direct interaction of IncV with VAP25, the interaction of STIM1 with the inclusion membrane by an unknown mechanism26, and the interaction of MrcA with the inositol triphosphate receptor (IP3R)17. STIM1 and IP3R localization to microdomains of the inclusion membrane26 demonstrate that regulators of Ca2+ homeostasis are recruited during chlamydial development. Therefore, we investigated if chlamydial development disrupts host cell Ca2+ homeostasis. Here we provide evidence that SOCE is impaired by the midpoint of the chlamydial developmental cycle and the SOCE-inducible NFAT/calcineurin signaling pathway is concurrently abrogated, which could have major implications on host cell physiology.The 2+ mobilization, the ratiometric Ca2+ indicator, Fura-2, AM was used to assess changes in host cell intracellular Ca2+ concentration ([Ca2+]i). HeLa cells either uninfected or infected with C. trachomatis L2 were loaded with Fura-2, AM at the desired time post infection. The binding of Ca2+ to Fura-2, AM induces a shift in its fluorescence excitation from 380 to 340 nm. Therefore, a 340 nm/380 nm fluorescence ratio of Fura-2, AM was used to determine relative changes in Ca2+ concentration. A Ca2+ re-addition assay27 was used to obtain ratiometric measurements in a resting state, during induced ER Ca2+ leakage, and throughout SOCE. Following a resting state baseline reading in a Ca2+-free Ringer’s solution, cells were incubated with Ca2+-free Ringer’s solution containing either thapsigargin (TG) or the vehicle control, DMSO. TG is an inhibitor of the SERCA Ca2+ pump responsible for mobilizing Ca2+ from the cytosol into the lumen of the ER or sarcoplasmic reticulum. TG thus causes an increase in cytosolic Ca2+ by impairing ER Ca2+ uptake while ER Ca2+ depletion occurs via passive ER Ca2+ leakage through ER translocon complexes29. When TG treated and DMSO control HeLa cells were moved to a Ca2+-containing Ringer’s solution, a distinctive increase in [Ca2+]i was detected in TG-treated, but not DMSO treated, cells indicating that Ca2+ depletion of the ER resulted in the activation of SOCE , early-to-mid- (8 hpi), mid- (24 hpi), and late- (46 hpi) cycle developmental time points  in infected cells at 8 h post-infection and 90.8% ± 2.7% in uninfected versus 93.7% ± 2.0% in infected cells  at 24 h post-infection. Viability dropped slightly in infected cells at 46 h post-infection from 88.0% ± 3.3%  in uninfected to 80.7% ± 1.9%  in infected cells (p < 0.013).Intracellular pathogens depend upon viability of the host cell to complete their intracellular development2+ at the cell population level. To assess the influence of C. trachomatis serovar L2 on host cell Ca2+ mobilization at a single cell level, we used the Ca2+ indicator Fluo-4, AM with live-cell microscopy to determine changes in [Ca2+]i in infected and uninfected cells. To quantify the normalized relative change in Fluo-4, AM fluorescence intensity (F) for each cell, ΔF/F0 was calculated. The baseline resting state fluorescence (F0) was the average of the first four mean intensity measurements in Ca2+-free Ringer’s solution, and ΔF = F − F0 was used to calculate the change in fluorescence.The Fura-2, AM-based method measured changes in cytoplasmic Ca2+]i mobilization in uninfected and C. trachomatis-infected cells was performed at a mid-cycle (24 h) developmental timepoint. In uninfected and infected HeLa cells treated with the DMSO carrier, stochastic Ca2+ elevations were observed in a small subset of cells, and when DMSO-treated control and infected cells were placed in Ca2+-Ringer’s solution, there was a modest increase in [Ca2+]i , GCaMP6m, was used to corroborate the results of the Fura-2, AM and Fluo-4, AM based quantification of [Ca2+]i mobilization in uninfected and C. trachomatis-infected cells. The GCaMP family of GECIs are single fluorescent protein indicators that operate by utilizing a circularly permuted GFP linked to calmodulin and the Ca2+-dependent calmodulin-interacting peptide M13 from myosin light chain kinase (MLCK). Ca2+ binding induces a conformational change in the circularly permuted GFP causing a large increase in fluorescence intensity30. Utilizing GCaMP6m, a relative change in GFP fluorescence can be used to determine changes in [Ca2+]i. C. trachomatis L2 expressing mScarlet permitted visualization of chlamydial inclusions during imaging. HeLa cells infected with mScarlet C. trachomatis L2 and transfected with pN1-GCaMP6m-XC were tested to assess Ca2+ mobilization.The genetically encoded CaC. trachomatis serovar L2 demonstrated that SOCE was suppressed in infected cells . Similar to the C. trachomatis L2 results, C. trachomatis D impaired SOCE of the host cell by the mid-cycle developmental timepoint -GFP plasmid. A pilot study indicated that the optimal time following TG treatment and Ca2+ incubation for NFAT-GFP nuclear translocation in HeLa cells was 18 min post-Ca2+ addition . No significant difference in NFAT-GFP N/C was observed for C. trachomatis-infected cells treated with TG post-Ca2+ incubation.  for each condition. The NFAT-GFP N/C was measured as the mean fluorescence intensity of nuclear NFAT-GFP / the mean fluorescence intensity of cytoplasmic NFAT-GFP for individual cells. Cells with an observable inclusion containing mScarlet on. Fig. b. To visell Fig. c. Collec2+ dynamics in C. trachomatis-infected cells and demonstrate that C. trachomatis inhibits SOCE of the host cell by a mid-cycle (24 h) developmental time point. This inhibition of SOCE was confirmed using three independent methods of intracellular Ca2+ quantitation. High concentrations of intracellular Ca2+ resulting from SOCE induction can act as a second messenger to activate numerous signaling pathways31. Among the pathways activated is the calcineurin/NFAT pathway. Sustained high concentrations of intracellular Ca2+ activates the phosphatase calcineurin which, in turn, dephosphorylates the cytoplasmic components of the NFAT transcription complex to trigger NFAT translocation into the nucleus32. NFAT transcription complexes regulate genes encoding immunomodulatory proteins or involved in developmental cellular differentiation34. C. trachomatis inhibition of SOCE had the downstream effect of inhibiting the calcineurin/NFAT pathway, thus providing a biological confirmation of the intracellular Ca2+ quantitation. The impairment of NFAT1 nuclear translocation demonstrate a specific signaling pathway that is affected by suppressing SOCE in C. trachomatis infected cells and likely has a multifaceted impact on host cell physiology and chlamydial pathogenesis.We examined Ca35. The cellular requirements for the two different release mechanisms are unique. Myosin II and Ca2+ are essential for chlamydial extrusion-based dissemination35. Ca2+ appears to be an important determinant for chlamydial release mechanism from the host cell, however, the suppression of SOCE indicates that other sources of Ca2+ must be utilized. The lack of synchrony and the fact that all infected cells do not undergo extrusion may present challenges in characterizing the formation of Ca2+ microenvironments near the inclusion microdomain.At the end of their developmental cycle, chlamydiae are released by one of two distinct mechanisms for infection of adjacent cells and subsequent cycles of infection. Infectious EBs are released either by lysis of the infected cell, or intact or partially intact membrane bound inclusions are released by a process known as extrusionC. trachomatis infected cells. STIM1 and IP3R are recruited to and enriched in microdomains on the inclusion membrane26. IP3R is recruited to the inclusion membrane through interactions with the chlamydial protein, MrcA17. ATP-induced IP3R activation in C. trachomatis infected cells37 suggests MrcA does not have an inhibitory effect on IP3R. The mechanism of STIM1 recruitment is unknown though STIM1 is known to form complexes with IP3R38. Although STIM1 is recruited to the chlamydial inclusion, Orai1 was not and remained localized to the host plasma membrane26. A model for possible interactions and events associated with inhibition of SOCE and NFAT translocation is depicted in Fig. Although the detailed mechanisms of chlamydial inhibition of SOCE remain unknown, at least two host components involved in calcium homeostasis are inappropriately localized in 39. Puncta formation was previously observed in cells overexpressing fluorescent protein tagged STIM1 and Orai126. While different pools of mCherry-STIM1 localized to the inclusion or the ER, Orai1-GFP did not associate with the inclusion membrane. When these cells were induced with TG to undergo SOCE, the inclusion membrane-associated mCherry-STIM1 remained at the inclusion, but the pool of ER-distributed mCherry-STIM1 demonstrated punctate staining indicative of ER-PM junctions necessary for the formation of CRAC channels. This suggested that an unknown chlamydial factor retains STIM1 at the inclusion membrane when CRAC channel formation is induced and likely limits the amount of available STIM1 for formation of these channels. Some caution must be exercised in interpretation of these results since overexpression of STIM1 or Orai1 can dramatically increase the number of CRAC channels40. The stoichiometry of STIM1 to the C. trachomatis sequestering agent will thus be important when assessing the influence of C. trachomatis on SOCE since STIM1 binding to the inclusion membrane presumably lowers the available STIM1 for binding Orai1 and could lower the endogenous STIM1:Orai1 molecular ratio. Re-analysis of the data from Dzakah41 revealed that none of the isoforms of STIM1 or Orai1 were significantly up- or down-regulated at 20 h post chlamydia infection. As to whether or not STIM1 expressed at endogenous levels are able to form puncta in infected cells remains unclear.TG treatment induces puncta formation and co-localization of Orai1 and STIM1 in uninfected cellsC. trachomatis infection for genital infections in women and ocular infections are the endocervical epithelium and conjunctival epithelium, respectively4. As the first targets of infection, mucosal epithelial cells act as first responders to pathogen challenge by the secretion of cytokines and chemokines, and thus play a key role in the innate immune response42. In an immortalized primary human endocervix-derived epithelial cell line, a productive C. trachomatis infection mitigated a pro-inflammatory cytokine and chemokine response. Although the mechanism was not defined, it was proposed that the circumvention of a robust cytokine and chemokine response represented a potential evasion strategy promoting the establishment of a favorable intracellular niche within the endocervix epithelium43. SOCE impacts cytokine and chemokine signaling events31. The influence of C. trachomatis-mediated suppression of SOCE warrants further investigation.The primary sites of 19. NFAT is also expressed in multiple cell types19, including epithelial cells46 and endothelial cells47, however, direct information on how disruption of NFAT signaling might impact pathogen interaction with epithelial cells is limited. African swine fever virus inhibits NFAT-regulated transcription of immunomodulatory proteins by synthesis of a protein, A238L, that directly binds to and inhibits the calcineurin phosphatase activity required for NFAT activation48. Reactivation of latent Epstein-Barr virus to a lytic state via a Ca2+/calcineurin dependent activation of NFAT by a complex mechanism has been proposed to represent a negative feedback loop involving a viral protein, Zta, that directly binds to and attenuates NFAT49. H. pylori has the capacity to influence NFAT activity either positively or negatively by the bacterial proteins CagA or VacA, respectively, in gastric epithelial cells46. VacA forms an anion selective channel in the plasma membrane50, which is believed to deregulate membrane depolarization and inhibit SOCE. Although the mechanism of SOCE inhibition differs, C. trachomatis also inhibits SOCE and subsequent NFAT nuclear translocation. NFAT is a transcriptional regulator that, upon translocation to the nucleus, induces expression of several genes important in innate immune responses51. Presumably, inhibition of SOCE by chlamydiae and the resultant inhibition of NFAT translocation blocks transcription of NFAT regulated genes. We hypothesize that this inhibition of SOCE and NFAT signaling promotes chlamydial survival, possibly by downregulation of chemokine or cytokine production. Further studies in human primary cervical epithelial cells or animal model systems will undoubtedly be necessary to fully elucidate the benefits to chlamydial and host survival.The impairment to calcineurin activation and NFAT1 nuclear translocation demonstrates a specific signaling pathway that is affected by chlamydial suppression of SOCE. Although the major functions of NFAT are often considered in innate immune cells such as lymphocytes, macrophages, dendritic cells, or neutrophils, NFAT plays multiple additional roles in development and cellular differentiation2 in a humidified incubator. The C. trachomatis D (UW-3-Cx) and L2 (LGV 434) serovars were cultured in HeLa cells and EBs purified by density gradient centrifugation as previously described52. Infectious titers were determined by Inclusion Forming Unit (IFU) assays53 with inclusions detected by indirect immunofluorescence. Viability of infected and uninfected cells was determined by Trypan Blue exclusion.HeLa 229 human cervical epithelial-like cells (American Type Culture Collection) were cultivated in RPMI-1640 (Gibco) supplemented with 5% fetal bovine serum (HyClone) at 37 °C and 5% CO4 cells/well in 96-well, black-wall, clear-bottom plates (Costar). Transfection complexes, either ON-TARGETplus Non-Targeting Control Pool siRNA (Dharmacon) or ON-TARGETplus SMARTpool STIM1 siRNA (Dharmacon) were diluted to a 1 µM working concentration in Opti-MEM. An equivalent volume of the diluted DharmaFECT 1 reagent and diluted siRNA were combined and incubated at room temperature for 30 min to form siRNA/DharmaFECT complexes. For the mock transfection, DharmaFECT 1 reagent in Opti-MEM was used. HeLa growth medium was exchanged with Opti-MEM while siRNA transfection complexes were generated. Following incubation, 0.1 mL of the diluted transfection complex was added per well. Cells were incubated for 48 h prior to cell infection.For siRNA transfections, HeLa cells were seeded at 1 × 10C. trachomatis, L2 for the stated times or uninfected were transfected with the desired mammalian expression vector using X-tremeGENE HP DNA transfection reagent (Sigma) according to the manufacturer’s instructions.For mammalian expression vector transfections, HeLa cells that were either infected with 4 cells/well. Cells were infected the following day with C. trachomatis serovar L2 at an MOI of 1. At the desired time post infection, medium was removed and cells were washed with PBS. Cells were loaded with 5 µM Fura-2, AM in Ca2+-containing Ringer’s Solution  for 1 h at room temperature. Cells were washed twice with PBS and incubated in Ca2+-free Ringer’s Solution  for 30 min at room temperature. Following incubation, Fura-2, AM readings were performed by measuring fluorescence at 340/11 nm and 380/20 nm excitation and 508/20 emission every 20 s for 5 min using a Cytation 5 (BioTek). The solution was replaced with Ca2+-free Ringer’s solution containing either 2 µM thapsigargin (TG) or an equivalent volume of DMSO (vehicle control). Fluorescence readings were repeated every 20 s for 5 min. Each solution was then exchanged with Ca2+-containing Ringer’s solution, and fluorescence readings were taken every 20 s for 5 min. Each condition was performed in triplicate.HeLa cells were seeded in 96-well, black-walled plates at a density of 2 × 104 cell/well in a 24-well, glass-bottom Cellvis imaging plate. The following day, HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 1. At the desired time post-infection, cultures were washed with PBS and loaded with 2.5 µM Fluo-4, AM in Ca2+-containing Ringer’s Solution for 1 h at room temperature. Cells were washed twice with PBS and incubated in Ca2+-free Ringer’s solution  for 30 min at room temperature. Following incubation, cells were imaged using the Nikon Ti2e with a CFI60 Super Plan Fluor Phase Contrast ADM ELWD 40 × Objective Lens . ND acquisition (NIS-Elements 64-bit version 5.11.02 software (Nikon) was set to acquire images from 4 locations per well for infected and uninfected condition. ND acquisition was programmed to image every 30 s for 5 min. At the end of the Ca2+-free buffer imaging, the Ca2+-free buffer was exchanged with Ca2+-free buffer containing either 2 µM thapsigargin (TG) or DMSO carrier. Cells were imaged for another 5 min imaging interval. At the end of that interval, buffers were exchanged with a Ca2+-containing Ringer’s solution , and images aquired for another 5 min interval. Huygens Essential software version 20.04 (Scientific Volume Imaging) was used to stabilize frames between time points and Imaris × 64 software version 9.6.0 (Oxford Instruments) was used to measure the mean fluorescence intensity of Fluo-4 per cell at each time point.HeLa cells were seeded at 2 × 104 cell/well in a 24-well, glass-bottom Ibidi imaging plate. The following day, HeLa cells were infected with C. trachomatis serovar L2 expressing mScarlet at an MOI of 1 and were transfected with pGP-CMV-GCaMP6m 4 h post-infection. The pGP-CMV-GCaMP6m plasmid was a gift from Douglas Kim and the GENIE project 54. At the desired time post infection, medium was removed from cells, and cells were washed with PBS. Cells were incubated in Ca2+-free Ringer’s solution for 15 min. Imaging, solution exchanges, image processing, and fluorescence measurements were performed as described in the Fluo-4, AM Ca2+ measurements section. Imaging was performed at 37 °C, 5% CO2, 92% relative humidity using a stage-top incubation chamber (Okolab).HeLa cells were seeded at 1 × 102+ measurement section, except that cells that were transfected with the HA-NFAT1(4-460)-GFP plasmid. The HA-NFAT1(4-460)-GFP plasmid was a gift from Anjana Rao 55. At the desired time post infection, cells were washed twice with PBS, and then incubated in Ca2+ -free Ringer’s solution containing 1 ug/mL Hoechst stain for 30 min. Buffer was then exchanged with either DMSO or 2 µM TG Ca2+-free Ringer’s solution. Immediately following treatment, an image was acquired using the Nikon Ti2e with a CFI60 Super Plan Fluor Phase Contrast ADM ELWD 40 × Objective Lens. ND acquisition was programmed to acquire images from 4 locations per well for each condition. After incubating cells with either DMSO or TG for 5 min, the solution was exchanged with the Ca2+-containing Ringer’s solution, and then imaged at 18 min post Ca2+ addition. Imaging was performed at 37 °C, 5% CO2, 92% relative humidity using a stage-top incubation chamber (Okolab). Following imaging, Huygens Essential software version 20.04 (Scientific Volume Imaging) was used to stabilize frames between time points and Imaris × 64 software version 9.6.0 (Oxford Instruments) was used to measure the mean fluorescence intensity of NFAT-GFP in the nucleus and the cytoplasm of individual cells before and after SOCE induction. The NFAT-GFP N/C ratio was calculated as the mean nuclear NFAT-GFP fluorescence intensity ∕ the mean cytoplasmic NFAT-GFP fluorescence intensity.Infections and transfections were performed as stated in the GCaMP6m CaC. trachomatis serovar E infected samples were accessed through the Sequence Read Archive (BioProject PRJNA666640). Paired reads were mapped to STIM1 (Gene ID 6786), ORAI1 , and nine housekeeping genes56 using the Geneious read mapper (Geneious Prime version 2022.1.1). Expression levels were calculated for each gene simultaneously with ambiguously mapped reads counting as partial matches and transcripts were compared across all samples normalizing to the median gene expression ratios using the DESeq2 method. Significant differences in expression were determined as greater than 1 or less than − 1 log2 fold change with a p-value of < 0.05.The RNAseq dataset from Dzakah et al.  was analyzed. The runs for uninfected or 20 hpi Statistical analysis was conducted using Prism version 9.1.1 software for Windows (GraphPad). An unpaired Student’s T test was performed for Fura-2, AM analysis, and Kruskal–Wallis test with Dunn’s posttest was performed for Fluo-4, AM relative change, GCaMP6m relative change, and NFAT-GFP N/C ratios. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.Supplementary Information 1.Supplementary Video 1.Supplementary Video 2.