Two types of scores were established to evaluate the abilities of the various methods to identify the most useful parameters/markers for distinguishing between AML blasts and normal myHPCs, as well as to distinguish between different AML groups. The Infinicyt Compass database-guided analysis was found to be a more user-friendly tool than other analysis methods implemented in the Cytobank software. According to the developed scoring systems, the principal component analysis based algorithms resulted in better discrimination between AML blasts and myHPCs, as well as between blasts from different AML groups. The most informative markers for the discrimination between myHPCs and AML blasts were CD34, CD36, human leukocyte antigen-DR (HLA-DR), CD13, CD105, CD71, and SSC, which were highly rated by all evaluated analysis algorithms. The HLA-DR, CD34, CD13, CD64, CD33, CD117, CD71, CD36, CD11b, SSC, and FSC were found to be useful for the distinction between blasts from different AML groups associated with recurrent genetic abnormalities. This study identified both benefits and the drawbacks of integrating multiple high-dimensional algorithms to gain complementary insights into the flow-cytometry data.Acute myeloid leukemias (AMLs) are a group of hematologic malignancies that are heterogeneous in their molecular and immunophenotypic profiles. Identification of the immunophenotypic differences between AML blasts and normal myeloid hematopoietic precursors (myHPCs) is a prerequisite to achieving better performance in AML measurable residual disease follow-ups. In the present study, we applied high-dimensional analysis algorithms provided by the Infinicyt 2.0 and Cytobank software to evaluate the efficacy of antibody combinations of the EuroFlow AML/myelodysplastic syndrome panel to distinguish AML blasts with recurrent genetic abnormalities ( The application of flow cytometry in the clinical evaluation of hematological diseases has undergone a constant evolution over time. Flow cytometric immunophenotyping is widely accepted as an indispensable method for the diagnosis and monitoring of lymphoid pathologies. In contrast, for hematological pathologies characterized by a wide heterogeneity of neoplastic cell population, such as the acute myeloid leukemias (AMLs), multicolor flow cytometry (MFC) is still considered a complementary tool in diagnosis and follow-up. Multicenter studies have reported relatively high numbers of false-positive cases when measurable residual disease (MRD) in AML was examined, resulting in a low specificity of 71%, even when using standardized protocols, likely due to differences in subjective interpretation . The EurThe manual processing based on Boolean gating of the high-dimensional dataset obtained in AML immunophenotyping is highly subjective, time-consuming, not easily scalable to a high number of dimensions, and is inherently inaccurate because it does not account for cell population overlap . TherefoThe automatic population separator (APS), canonical correlation analysis (CCA), neighbor-APS (NAPS) diagrams based on PCA algorithm and t-distributed stochastic neighbor embedding (t-SNE) based on K-means clustering algorithm are implemented in the Infinicyt software developed by EuroFlow (EF) to allow comparison of cell populations from different case groups with a database .Citrus software also allows detection of differences between groups and correlate or predict experimental outcomes . In addiOther algorithms for automatic gating and 2D mapping of high-dimensional datasets have been developed, such as spanning-tree progression analysis of density-normalized events SPADE;  and t-SNMLL (KMT2A) gene alterations. Data were acquired in a standardized manner and were analyzed using the Compass algorithm in the Infinicyt software and the Citrus, viSNE, and SPADE algorithms from the Cytobank software.The identification of algorithms useful for the unsupervised analysis of MFC data in AML is the first step in developing automated data analysis tools. This retrospective study aimed to evaluate the usefulness of novel MFC data analysis software for comparing the immunophenotypic profile of myeloid hematopoietic precursors (myHPCs) from normal bone marrow (NBM) samples with that of leukemic blasts from AML cases carrying recurrent genetic abnormalities, such as t, t, inv(16), and Here, we present some advantages and disadvantages of different unsupervised MFC analysis tools for evaluating the performance of the EF AML/myelodysplastic syndrome (MDS) antibody panel for (EF AML/MDS panel), for the identification of the most useful markers for separating AML blasts from their nearest normal myHPCs counterpart, and for the identification of the AML group-specific phenotypic imprints.RUNX1/RUNX1T1 AML, 13 patients with t PML/RARα AML, eight patients with inv(16) or t CBFB/MYH11 AML, and 11 patients with MLL/KMT2A gene-altered AML (MLL AML), diagnosed at the Institut de Cancérologie Lucien Neuwirth  between 2013 and 2017, were analyzed and compared with data from 23 NBM samples  from the University Hospitals of Saint-Étienne and Clermont-Ferrand, France.Samples from 39 AML patients, comprised of seven patients with t Written informed consent was obtained from each patient and healthy donor, as approved by the institutional procedures of the independent Ethics Committee and the Comité de Protection des Personnes – Île-de-France (NCT03233074/17.07.2017).The patients’ characteristics are summarized in 6 cells (106 per tube) were stained with the first three EF AML/MDS panel antibody combinations .At least 500,000 total events were acquired by tube using three FACS Canto II cytometers  in two university-affiliated hospitals in France (Saint-Étienne and Clermont-Ferrand). Data were analyzed using Infinicyt 2.0 software  and the software packages including the algorithms Citrus, viSNE, and SPADE from the Cytobank + monocytes (Tube 2), and CD10+ hematogones (Tube 1) , and CD45 , 3C, 4C (Tube 1) , 3C, 4C.Using this strategy, the initial bulk of myHPCs included the very immature CD117+ CD34+ precursors and the more mature CD117+ CD34− myHPCs from each of the three myeloid lineages.Regarding AML cases, this strategy allowed discrimination of both immature blasts (CD34+ CD117+), and those more matures (CD34− CD117+); the percentages of the blasts identified by MFC based on this strategy corresponded to those identified in morphology see .Subsequently, events corresponding to myHPCs and AML blasts were exported to new FCS files and analyzed using the Compass tool from the Infinicyt 2.0 and in Citrus, viSNE, and SPADE from Cytobank software. The ArcSinh-hyperbolic transformed data using a co-factor of 150 were subjected to analysis with the three listed algorithms from Cytobank for the following markers . In Infinicyt software, the negative visibility is by default corrected when the Logical scales are used.The all markers from the tubes, including the three markers of the backbone used for the pre-gating of AML blasts and myHPCs, have been used in analysis in order to identify potential differences in their intensity of expression.MLL AML.In the Infinicyt software, a database was built for each of the three myeloid lineages: neutrophilic (Tube 1 database), monocytic (Tube 2 database), and erythroid (Tube 3 database). The final databases for neutrophil, monocytic, and erythroid lineages contained cases belonging to five groups: NBM , t AML, t AML, inv(16) AML, and In accordance with EuroFlow recommendations , the filFour FCS files from samples stained with Tube 1 and Tube 2 antibody combinations and three FCS files for Tube 3 were excluded from the databases for the NBM group. These files showed >2.5 SD discordances compared with the group median for the markers used to select myHPCs and for the side scatter (SSC) values of the internal control populations, lymphocytes, neutrophils, and NRCs .MLL AML group . Therefore, one FCS file was excluded from the neutrophil lineage database for the t AML group, one FCS file was excluded from all databases for the t AML group, and one FCS file was excluded from the neutrophil and monocytic lineage databases for the ML group , 4A,B.Each group of cases was subsequently plotted in a balanced principal component analysis (PCA) plot with medians for each patient and SD curves for each group of cases to compare each case with each of the five groups and for each of three myeloid lineages. The markers that received a weight over 10% in the first or second principal component in an APS view of the AML blasts and the nearest normal myHPCs were considered useful for distinguishing between groups . These rFor the configuration of Cytobank analysis software  and data interpretation, we used the practical guide published by 1 association model; (6) cluster characterization of medians; (7) equal event sampling; (8) 5,000 events sampled per file; (9) 5% minimum cluster size (Cytobank default); (10) five-fold cross-validation (Cytobank default); and (11) 1% false discovery rate (Cytobank default).The FCS files containing the manually gated myHPCs and AML blasts were imported into Cytobank and then subjected to Citrus analysis using the following settings: (1) singlet, non-debris cell as input; (2) hierarchical clustering in order to group cells into populations; (3) analysis of 10 out of 12 possible parameters per tube  target number of nodes = 20; and (2) 100% percent downsampling. Analysis was performed using 10 of 12 possible parameters per tube , and no fold change calculations were made for this data set.Spanning-tree progression analysis of density-normalized events trees were initially visually investigated to identify nodes that were potentially different in cellular abundance between conditions. Two SPADE runs of datasets were performed. Clusters organization by analyzed group (AML or NBM) were not conserved between runs, resulting in considerable differences between the SPADE tree from the two runs , and ren2 and then subjected to viSNE analysis.Manually gated singlet events corresponding to myHPCs in NBM and to AML blasts were imported into CytobankAccording to previously published data , viSNE wviSNE clustering analysis was performed on 10 of 12 possible parameters . Equal event sampling was selected at 100,000 events per individual . The advanced settings were set as per the Cytobank default settings: random seed, 1,000 iterations, perplexity = 30, theta = 0.5.Data accompanying each viSNE node were downloaded from Cytobank to calculate the frequency of events in each node, followed by testing for statistical significance.The nodes that showed the most statistically significant differences in the cellular abundance between groups  and Kruskal–Wallis tests for multiple comparisons.The distribution of variables was measured using one-tailed paired and unpaired TM  was used to compare the differences in markers expression between AML blasts from different groups and myHPCs as reflected by viSNE algorithms.The identification of significant nodes in the cellular abundance between groups was determined using Microsoft Excel 2019 . The Kruskal–Wallis multiple comparative test from GraphPad Prism 5A scoring system was developed for identification of the most useful markers for discrimination between the AML blasts and the normal myHPCs and for testing the capacity of different methods for their identification . The assIn Infinicyt software, the differences between AML blasts and normal hematopoietic progenitor cells (HPCs) were visualized in separate, non-fixed APS plots using all eight markers and the FSC and SSC parameters of each tube in line with previously published studies . The relFor the other software, the differences in the intensity of markers on the populations of interest was assessed on the scales of the plots provided by the different software, after the arcsine transformation of the data, which allow a normal distribution of the data and improves the representation of cells with minimal fluorescence, as it was demonstrated by Citrus boxplots were used to evaluate differences between the analyzed markers and parameters. Differences in markers expression and FSC, SSC between AML blasts and normal HPCs > 0.5 between AML blasts and normal HPCs received 2 points, those inferior to 0.5 received 1 point and the absence of difference was noted with 0 points.Logarithmic differences were used to interpret markers and parameter differences with viSNE. Differences in markers expression and FSC, SSC between AML blasts and normal HPCs > 1 log received 2 points; those between 0.5 and 1 log, 1 point and the absence of difference was noted with 0 points.The total score to evaluate the discriminative capacity of each marker to distinguish between AML blasts and normal myHPCs was obtained by adding all values obtained by the respective marker or parameter for distinguish between each AML group and normal HPCs and for each analysis method. The threshold score for marker relevance was >10.MLL AML versus normal myHPCs].The total score used to evaluate the effectiveness of each algorithm for the ability to distinguish AML blasts from normal myHPCs was obtained by summing the values obtained of each two-by-two paired group   AML versus t AML, t AML versus inv(16) AML, t AML versus MLL AML] .We sought to evaluate whether the selection of myHPCs and AML blasts using primarily the backbone markers present in Tubes 1–3 of EF AML/MDS panel would allow appropriate gating and discrimination between normal myHPCs and AML blasts, as well as between blasts from different groups of AML cases.MLL AML – were used in this study on assumption that they might constitute homogeneous groups in terms of immunophenotype.The most common subtypes of AML with recurrent genetic abnormalities – AML t, AML t, AML inv(16), and We used two clustering algorithms, Citrus and SPADE, and two dimensionality-reduction methods, viSNE, and a PCA-based algorithm  to explore the immunophenotypic features associated with AML blasts compared with those associated with normal myHPCs.Generalized reduction in markers expression heterogeneity was observed on the cells of the nodes containing a significantly increased number of events in AML cases compared to normal myHPCs when viSNE algorithms were used, as reflected in dot plots –4B–D. HoIn line with previously published studies  we used However, these algorithms can help to identify which are the most informative markers in distinguishing AML blasts from their nearest normal myHPCs counterparts. This step is useful for improving the antibody combinations and the gating strategy for myHPCs or AML blasts.Based on the PCA analysis (Infinicyt software) and the analysis of clusters with the minimum prediction error (Citrus software), the FSC and SSC parameters may be useful for distinguishing between AML blasts and normal myHPCs. SSC seemed to be more useful for this purpose, and both algorithms highlighted the SSC increase in t AML blasts , 6B, altAlthough included in the analysis, the viSNE algorithm did not indicate that FSC and SSC parameters could be useful for separating AML blasts and normal myHPCs  AML blasts vs. myHPCs (P = 0.0196) and in inv(16) AML blasts vs. myHPCs (P = 0.0205), but a significant decrease in t AML blasts vs. myHPCs (P = 0.0152) and in MLL AML blasts vs. myHPCs (P = 0.0102).Citrus boxplots corresponding to the parent nodes from the hierarchy , the parThe power of discrimination of CD34 seemed to be potentiated by an association with other antibodies, nearly always resulting in much better discrimination in two of the three tubes (Tubes 1 and 2), as reflected by the Compass algorithm (Infinicyt) . AbsenceMLL AML blasts (P = 0.0439) and in inv(16) AML blasts vs. myHPCs (P = 0.0405), and a significant decrease in t AML blasts vs. myHPCs (P = 0.0352), but not in MLL AML blasts vs. myHPCs (P = 0.0697). A possible explanation that no statistical significance was obtained for the intensity of expression of HLA-DR between MLL AML blasts and normal myHPCs is likely related to the important heterogeneity of expression of this marker between the different MLL AML cases, as reflected by Infinicyt histograms  AML , 3B–D anL blasts , with inL blasts , 2C and L blasts , 4B. Whestograms .MLL AML blasts compared with normal HPCs with the combination of markers from the third tube of EF AML/MDS panel  AML, inv(16) AML blasts and slightly decreased on the t AML, DS panel . This obDS panel ].MLL AML (MLL AML) .In addition, the Infinicyt histograms revealed that CD117 could be increased also in some cases of MLL AML  and by tMLL AML) . HoweverCD45 was the least discriminant of the backbone markers and was slightly more intensely expressed by the t AML and inv(16) AML blasts compared to normal myHPCs, as highlighted especially by Citrus algorithms in Tube 3 .x-axis) or second  principal component generated by the Infinicyt algorithms (Table T1 from P = 0.0217), but also between MLL AML blasts (decreased expression) compared with their nearest normal myHPCs (P = 0.0429).The tables showing the contribution of each parameter to the first  AML and t AML blasts (decreased expression) and normal myHPCs , 2B, andMLL AML blasts than by normal myHPCs  showed that CD64 was somewhat discriminant, identified as being slightly more intensely expressed by t AML and l myHPCs .According to analysis with the viSNE algorithm, the CD300e (IREM2) marker showed increased expression on t AML blasts, and the CD14 and CD35 markers showed decreased expression on the leukemic blasts from the AML inv(16) group, compared to the normal myHPCs in the nodes with statistically significant increased numbers of events in AML cases. However, the parameter band histogram from Infinicyt did not show a notable difference for CD300e on t AML blasts, nor for CD14 on inv(16) AML blasts compared to normal myHPCs .The CD36, CD105, and CD71 from Tube 3 were ranked by Compass-based analysis (Infinicyt) among the discriminating markers between AML blasts and normal myHPCs –4A. viSNOverall, the erythroid lineage-associated antigens CD36, CD105, and CD71 have a reduced expression on the AML blasts compared to normal myHPCs  row T3. The relative contribution that each marker or parameter to distinguish between the blasts of different AML groups and normal myHPCs was scored based on weights in the first or second principal component in an APS diagram in Compass (Infinicyt), on Citrus boxplots (Cytobank) and based on logarithmic differences observed for median fluorescence intensity (MFI) with viSNE (Cytobank). The total score to evaluate the discriminative capacity of each marker to distinguish between AML blasts and normal myHPCs was obtained by adding all values on each line. The threshold score for marker relevance was >10. Compass-guided analysis showed that the best separation capacity was achieved between inv(16) AML blasts and normal myHPCs (score = 18), thereafter the separation between by t AML blasts and normal myHPCs (score = 15), followed by the separation between t AML and MLL AML blasts and normal myHPCs (score = 13).The total score used to evaluate the effectiveness of each algorithm for the ability to distinguish AML blasts from normal myHPCs was obtained by summing the columns values.Based on this score , the ComAs revealed by all analysis algorithms, in descending order, the following seven parameters were the most useful to distinguish between AML blasts and normal myHPCs: CD34; CD36; then with equal weight HLA-DR, CD13, CD105, and CD71; and finally SSC .MLL AML], and weak discrimination between inv(16) AML and MLL AML blasts with the markers from the Tubes 1 and 2. The most useful markers in separating the blasts from t AML and inv(16) AML groups were CD34, HLA-DR, and the SSC parameter, followed by FSC and the markers CD64, CD71, CD36, and CD33  AML from those of the rameters .Database-guided analysis in the Infinicyt software allowed a rapid analysis of the phenotypic differences between the blasts from different AML groups.The immunophenotypic characterization of AML blasts by MFC is standard of care in AML diagnosis and follow-up. However, there is no broad consensus on the panel of antibodies to be used or on the algorithms of analysis that would allow for the standardization of practices at the international level.The EuroFlow Consortium, a European collaborative research group has developed and validated a eight-color EF AML/MDS panel that allows the detection of AML blasts, their lineage assignment, and the accurate evaluation of the maturation profiles of myeloid lineages . HoweverThis preliminary, retrospective study was performed to evaluate the different tools provided by different software for data analysis to evaluate whether the first three tubes of the EF AML/MDS panel could be used for discrimination between myHPCs and AML blasts using this panel or its utility for AML blast classification in different cytogenetic groups.The different methods analysis had a range of advantages and disadvantages.The major advantage of the Citrus algorithm (Cytobank) is the capacity to build predictive models that allows identification of the minimum set of biomarkers necessary to discriminate effectively between the groups and estimation of the accuracy with which these biomarkers can do this .Of the others Cytobank algorithms, the SPADE analysis provides information about the population structure and the heterogeneity of population events, and the viSNE tool aids with the identification of distinct phenotypes between the groups of cases.However, the software on the Cytobank platform is less practical for multicenter studies because it does not allow overlapping data with different nomenclature for antibodies or fluorochromes which is frequently observed in these types of studies.The SPADE software allows the creation of population trees reflecting the size of cell clusters and the relationships between them. In agreement with other published data, a major drawback of the SPADE interface is the loss of single-cell resolution , but alsThe viSNE software from Cytobank has also a disadvantage; the configuration of this software is operator-dependent. In particular, the operator sets the number of iterations, which specifies the number of runs of the algorithm, and the perplexity parameter, which influences the results.Another limitation of the viSNE tool was the difficulty we encountered when attempting to compare groups of cases, for example in visualization of differences of markers’ expression using heatmaps. When the number of parameters to be analyzed is great or the number of cases to compare is increased, the heatmap visualization and interpretation are difficult.Infinicyt 2.0 also allows the evaluation of the relationship between different cell clusters from a sample, or between different groups based on two new diagrams, the robust curve and population burst . HoweverIn Infinicyt 2.0, several new diagrams have been recently developed, such as t-SNE, Canonical Correlation Analysis (CA), Neighborhood Automatic Population Separator (NAPS), and Compass VI (CVI 1) diagrams, that also allow exploration of population heterogeneity. These are not expert-based decisions, and are therefore not associated with a significant component of individual subjectivity .The major advantage of the Infinicyt software is the possibility to perform the database-guided analysis. Compass-guided method is a rapid analysis tool, allowing comparison of the data acquired across different flow cytometry platforms, which was specially designed for a better visualization when comparing groups of cases. This method gives information about the parameters that are useful for separation of the groups, and therefore, can help to improve the combinations of antibodies. The Compass database-guided analysis based on PCA algorithm has proven its advantage in various studies, such as for acute leukemia orientation , B cell Our preliminary data show that the Compass database-guided analysis is a helpful tool for the identification of DfN patterns that may advance actual methods of minimal residual disease evaluation by MFC in AML, which are frequently based on the detection of leukemia-associated immunophenotypes (LAIPs) identified at diagnosis.Based on EF experience, the unequivocal identification of blast cells in BM samples is possible using solely the backbone markers from the EF AML/MDS panel . The uselow CD117+ CD34± HLA-DRvariable (low to high) population with undesirable mature cells.However, our data revealed that a strategy based solely on the expression of these markers cannot be used to clearly isolate the AML blasts with recurrent genetic abnormalities or normal myHPCs, due to the inability to avoid the contamination of the CD45Therefore, evaluating the capability of other markers for the distinction of AML blasts from normal myHPCs counterparts may be useful.CD38 appears to be a very useful marker, as the 92% of AML blasts express this protein, as reported by The data from our current study showed that the inclusion of myeloid lineage-associated markers, such as CD13, CD64, CD36, CD105, CD71, and CD33, in the gating strategy can increase the discriminatory power for distinguishing between AML blasts and normal HPCs that are committed toward different myeloid lineages.In line with this observation, Webber et al., noticed that second to CD38 expression, AML blasts most frequently expressed the myeloid lineage markers CD13 (91%) and CD33 (87%) .The CD64 is an useful marker for the distinction of early monocytic precursors, whereas CD105, CD36, and CD71 are useful for the identification of myeloid precursors that have committed to the erythroid lineage .The Compass database-guided AML analysis (Infinicyt) is a user-friendly interface, facilitating the rapid evaluation of differences between blasts from different types of AML. The identification of phenotypic imprints associated with AML groups with recurrent genetic abnormalities may allow for the detection of residual leukemic blasts, even in the absence of phenotypic identification at diagnosis.MLL AML blasts, the most important differences were observed for CD34, CD117, HLA-DR, CD13, CD11b, CD64, CD36, CD105, CD71, and CD33, in addition to the FSC and SSC parameters  AML group, PCA did not result in the clear distinction between AML blasts from varying groups, primarily due to the heterogeneity of marker expression across blasts harboring the same recurrent genetic abnormality. For example, the t AML group displayed large variability in the expression of CD34, HLA-DR, and CD13, whereas, for rameters .MLL AML groups represents the primary limitation of this study because these small sample sizes did not allow for the identification of possible subtypes inside these immunophenotypic heterogenous groups.The reduced number of cases included in the t AML and However, this preliminary study has allowed re-designing a 12-color panel combining backbone markers from the EF AML/MDS panel with the myeloid lineage-associated markers, such as CD13, CD64, CD36, CD105, CD71, and CD33 that could improve the discriminatory potential between myHPCs and AML blasts. This 12-color panel should be initially tested on homogenous phenotypic groups of AMLs  or AMLs having a well-established molecular target to be able to compare MFC data with molecular biology results in different time points of AML follow-up. Multicenter studies using the Compass database-guided analysis based on PCA algorithm is required for its validation. To this end, the following steps should be followed: the use of standardized protocols for sample preparation, and data acquisition, effective harmonization of the instruments between different centers, intra-center normalization of the instrument(s) using the daily 8 Peak Beads QC, correction of the compensations for the data files, correction of the mean fluorescence intensities of the cell surface markers to adjust the median values and eliminate the antibody batches variations between instruments [i.e., using a Python script, as previously described by Differences observed in the results obtained with these two types of software, Infinicyt and Cytobank maybe are related to the different development directions of the two packages. Cytognos strive efforts in the clinical classification functions, whereas Cytobank is focused on development of multidimensional analysis tools like tSNE and SPADE.The PCA-based algorithms can be efficiently used to analyze data with low dimensionality, but heterogenous. However, the results obtained depends on the discriminatory capacity of the antibody combinations used.In addition, high-dimensional analysis algorithms may help visualizing the phenotypic changes of the AML blasts under the pressure of microenvironmental factors and to explore the immune system disorders contributing to the AML onset and progression. Recent studies bring to front also the role of the interaction between bone, immune and cancer cells in protecting the latter from immune system attack . Data geIn conclusion, the new tools of data analysis in MFC: (1) avoid data misinterpretations; (2) allow study of well-defined population groups; (3) highlight useful markers for better discrimination between different clinical groups; and (4) enhance our understanding of the utility of the panels used in routine practice.The benefits of using these analysis tools are becoming increasingly evident, as evidenced by the growing number of studies that use them.https://flowrepository.org/, FR-FCM-Z3JL.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Comité de Protection des Personnes – Île-de-France (NCT03233074/17.07.2017). The patients/participants provided their written informed consent to participate in this study.C-MA: conceptualization, formal analysis, data curation, and writing – original draft preparation. C-MA, RV-M, LR, and JS: validation. ET and DG: clinical investigation. C-MA, RV-M, LR, JS, ET, LC, ED, and DG: writing – review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Autophagy is a cellular self-eating process that uses lysosome, the waste disposal system of the cell, to degrade and recycle intracellular materials to maintain cellular homeostasis. Defects in autophagy are linked to a variety of pathological states, including cancer. Calcium is an important cellular messenger that regulates the survival of all animal cells. Alterations to calcium homoeostasis are associated with cancer. While it has long been considered as cellular recycling center, the lysosome is now widely known as an intracellular calcium store that regulates autophagy and cancer progression by releasing calcium via some ion channels residing in the lysosomal membrane. In this review, we summarize existing mechanisms of autophagy regulation by lysosomal calcium channels and their implications in cancer development. We hope to guide readers toward a more in-depth understanding of the importance of lysosomal calcium channels in cancer, and potentially facilitate the development of new therapeutics for some cancers.2+ is pivotal intracellular messenger that coordinates multiple cell functions such as fertilization, growth, differentiation, and viability. Intracellular Ca2+ signaling is regulated by both extracellular Ca2+ entry and Ca2+ release from intracellular stores. Apart from working as the cellular recycling center, the lysosome has been increasingly recognized as a significant intracellular Ca2+ store that provides Ca2+ to regulate many cellular processes. The lysosome also talks to other organelles by releasing and taking up Ca2+. In lysosomal Ca2+-dependent processes, autophagy is particularly important, because it has been implicated in many human diseases including cancer. This review will discuss the major components of lysosomal Ca2+ stores and their roles in autophagy and human cancer progression.Ca The lysosome is an acidic single-membrane organelle first discovered in 1955 by Christian de Duve while investigating the mechanism of action of insulin ,3. It co2+ and calmodulin (CaM) dependent serine/threonine protein phosphatase, and translocates to the nucleus to promote the transcription of the CLEAR element, which subsequently promotes autophagy-lysosome pathway as well as exocytosis and phagocytosis  [TRPML1 is a CaP2] ,44 but iP2] ,45. NormP2] . TRPML1 P2] ,47 and sP2] ,48. DefiP2] .+/Ca2+ channels that can be evoked by both NAADP and PIP2 [2+ release to regulate autophagy [Two-pore channels, including TPC1 and TPC2 in human, are widely expressed in the endolysosomal system. They form a homodimer, with each pore-forming subunit comprising two repeats of six-transmembrane domains D,E. FuncIP2 ,62,63,64IP2 . TPCs arutophagy ,66 , a Ca2+-permeable non-selective cation channel found in somatosensory neurons [2+ permeable non-selective cation channel gated by ADP-ribose and Ca2+ [2+ channels (VGCCs), Ca2+ channels regulating the fusion of autophagosomes with lysosomes in neurons [2+ channels involved in the autophagy are TRPMLs and TPCs channels.Several other Ca neurons ; transieand Ca2+ ; snd P/Q neurons . However2+ after release. The refilling of lysosomal Ca2+ store seems to be dependent on the lysosomal H+ (2+ store [2+ store is maintained through the Ca2+-H+ exchanger (CAX) in the vacuoles of yeast and plant, which is from some respects the equivalent of lysosomes in animal cells [2+/H+ exchanger may take part in the Ca2+ uptake in the lysosome [2+ release, manipulation of lysosomal pH may affect lysosomal Ca2+ concentration or its measurement. For example, because the lysosome contains substantial Ca2+ buffers [2+ much better at neutral pH [2+ without necessarily triggering lysosomal Ca2+ release and affecting total Ca2+ content [2+-binding affinity (Kd) of Ca2+ dyes sequestered in the lumen, because Kd drops significantly when lysosomal pH increases [2+ homeostasis by regulating membrane fusion and fission between compartments containing different amounts of Ca2+, H+, and their buffers [2+ refilling under more physiological conditions, Garrity et al. [2+. They reported that inhibition of the H+ gradient in the lysosome, for example, by V-ATPase inhibitors, did not affect Ca2+ refilling. Therefore, the identity of the channel or exchanger mediating lysosomal Ca2+ uptake is still unclear [2+ uptake into lysosomes is mediated by a low-affinity Ca2+ transporter [2+ entry [2+ into lysosomes is regulated by H+ gradient across the lysosomal membrane remains unclear.Due to the storage capacity, the lysosome needs to be refilled with Casomal H+ , because2+ store ,36,46;  formation of ER-lysosome membrane contact sites (MCS) [2+ transport from the ER to the lysosome through functional ER–lysosome membrane contact sites.ER Caefilling . Moreoveefilling ,76: (1) es (MCS) ,77,78,792+, the lysosomes, along with the ER, modulate cytosolic Ca2+ signaling events. On the one hand, lysosomal Ca2+ release can induce Ca2+ release from the ER using a mechanism similar to the Ca2+ induced Ca2+ release (CICR). This further causes Ca2+ entry from the extracellular space due to the depletion of the ER Ca2+ store, evoking global Ca2+ signals in human cells [2+ release are involved in this process. On the other hand, increasing evidence has suggested that, when lysosomal Ca2+ store is depleted, Ca2+ in the ER is transported to the lysosome via IP3R to refill the lysosome with Ca2+ [2+ signaling between the lysosome and the ER [2+ buffer to shape extracellular Ca2+ entry and the ER Ca2+ re-uptake [By releasing and taking up Caan cells . Both TRan cells  and TPC2an cells ,83,84-meith Ca2+ ,85. The d the ER  has alsod the ER ,87  genes, which were originally identified in yeast. The products of these ATG genes, together with other autophagy-related factors, regulate autophagosome formation, tethering, and fusion with lysosomes ,109,110.utophagy .Studies of mammalian systems have highlighted many important roles of autophagy in health and diseases including cell growth  and diff2+ can regulate autophagy, while mechanisms differ depending on the conditions. The interplay between lysosomal Ca2+ signal and autophagy has also been reported. In line with this, several lysosomal Ca2+-permeable channels have been suggested to regulate autophagy [It is widely accepted that Cautophagy ,120,121.2+ release channel in the lysosomal membrane, TRPML1 deficiency leads to defective autophagy including accumulation of autophagosomes and aggregation of p62 proteins [As a key Caproteins ,124,125.proteins , (2) ALGproteins , (3) proproteins , (4) Sytproteins ,128, (5)proteins ,23, and proteins   are found in the plasma membrane of excitable cells, P/Q-type VGCCs are recently reported to be expressed in lysosomes of both mice and fruit flies. Loss of VGCC leads to defects in autophagosome–lysosome fusion, indicating an important role of Ca2+ flux through this channel in autophagy [Although most voltage gated Cautophagy .2+ regulatory membrane proteins including Ca2+ channels, transporters, or Ca2+-ATPases [2+ channels play an important role in autophagy, the role of lysosomal Ca2+ channels in cancer development has attracted great attention in recent years [2+ signaling is a culprit in malignant tumor development [2+ signaling underlies several cancer hallmarks involving proliferation, metastasis, and angiogenesis and contributes to multidrug resistance in cancer therapy [2+ permeable channels, TRPMLs and TPCs, in cancer.An increasing number of tumorigenic pathways have been associated with an altered expression level or abnormal activation of Ca-ATPases ,140,141.-ATPases ,145,146.nt years ,148,149.elopment . Indeed, therapy ,159,160.2+ passing through TRPML1 is required for mTORC1 activation and cell growth [HRAS mutations [Recently, three groups have suggested independently that TRPML1 is required for tumor progression. Jung et al.  showed tl growth ,23. In Tl growth , TRPML1 l growth ,163,164,l growth ,163,165 l growth  suggestel growth  and cancutations . Future Notably, the clinical course of patients with cancer is also related to the TRPML1 expression level. Elevated TRPML1 expression level is reported to associate with the poor prognosis of pancreatic ductal adenocarcinoma (PDAC) patients. Overall survival rate and recurrence-free survival are significantly lower in patients with high TRPML1 expression as compared with patients with low TRPML1 expression .2+ release or silencing TRPML1 abrogated these effects. Loss or reduction of TRPML1 transcripts strongly correlates with short survival in glioblastoma patients, suggesting that the reduction of TRPML1 expression may be negatively linked to prognosis for glioblastoma patients [Although TRPML1 is often upregulated in cancer cells to promote cancer generation in glioblastoma TRPML1 agonist, MK6-83, reduce cell viability, and promote caspase-3-dependent apoptosis. Blocking TRPML1 dependent Capatients . TherefoTRPML2 has been linked to glioma, because a high level of TRPML2 was detected in glioma tissues, and because its expression level increased with pathological grades . MechaniThe role of TRPML3 in cancer development has not been well studied. Wu et al.  reported2+ release in melanoma cells [2+ release through NAADP/TPC2 to regulate angiogenesis [2+ signaling pathway in both tumor cells and endothelial cells.Emerging evidence has also suggested a role of TPCs in cancer development. Early studies suggested that TPC2 expression is increased in oral squamous cell carcinoma cell lines . Later sma cells . In agreogenesis ,178. Alt2+ release mobilizes ER Ca2+ via IP3R, subsequently promoting the proliferation of metastatic colorectal cancer (mCRC) cells by activating ERK and the PI3K/AKT signaling pathways [An earlier study showed that TPC1 transcripts are significantly higher than TPC2 transcripts in the SKBR3 human breast cancer cell line, highlighting the link between TPC1 expression and tumorigenicity . Indeed,pathways .2+ store. Given that Ca2+ signaling participates in processes that are important in cancer progression, such as autophagy, cell proliferation, growth and invasiveness, the tumor microenvironment, and resistance to anticancer therapies, it is therefore not surprising that exploration of the molecular mechanism underlying lysosomal Ca2+ homeostasis could be of great help in expanding our knowledge of the role of Ca2+ homeostasis in cancer development. In line with this, emerging evidence has suggested that Ca2+ release through lysosomal ion channels, the major lysosomal Ca2+ channels TRPML1 and TPC2, has been implicated in the progression of numerous cancers by controlling the autophagy pathway. Though more studies are needed to build a clear relation between lysosomal Ca2+ and cancer development and to underpin the precise mechanisms underlying the role of lysosomal Ca2+ in different types of cancers, developing potent and specific compounds targeting TRPML1 [It becomes evident that the lysosome acts as an important intracellular Cag TRPML1 ,153 and g TRPML1  could be


Some evidences indicate that this signalling pathway plays an extremely important role in bone formation and bone pathophysiologic changes. This review discusses the role of Ca2+/Calcineurin/NFAT signalling pathway in the process of osteogenic differentiation, as well as the influence of regulating each component in this signalling pathway on the differentiation and function of osteoblasts, whereby the relationship between Ca2+/Calcineurin/NFAT signalling pathway and osteoblastogenesis could be deeper understood.The bone remodelling process is closely related to bone health. Osteoblasts and osteoclasts participate in the bone remodelling process under the regulation of various factors inside and outside. Excessive activation of osteoclasts or lack of function of osteoblasts will cause occurrence and development of multiple bone‐related diseases. Ca 2+ in osteogenic precursor cells, thereby affecting the nuclear metastasis of NFAT and the process of osteogenic differentiation.Non‐classical Wnt signalling pathway, Orai1/STIM1, L‐VGCC and NCX can regulate the activity of CaM and CaN by modulating the concentration of Ca The bone remodelling process will last a lifetime to prevent the accumulation of micro‐damages in bone. Osteoclast‐led bone resorption and osteoblast‐led bone formation are tightly coupled in bone remodelling under various physiological and pathological conditions and can be affected by a variety of inside and outside factors such as hormones, cytokines, mechanical forces, magnetic fields, etc.2+ concentration, including calcineurin/nuclear factor of activated T cell (CaN/NFAT),2+/calcineurin/nuclear factor of activated T‐cells (Ca2+/CaN/NFAT) signalling pathway was originally discovered in T cells,2+/CaN/NFAT signalling pathway plays a more significant role in the regulation of cell growth and development, such as regulating cardiomyocyte differentiation, chondrocyte differentiation, myocyte hypertrophy, angiogenesis, myogenesis, skeletal development and regeneration, etc.2+/CaN/NFAT signalling pathway plays an extremely important role in affecting bone resorption, bone formation and bone physiopathological changes. Unlike the role of Ca2+/CaN/NFAT signalling pathway in osteoclast differentiation and bone resorption, which has been thoroughly discussed, the influence of this signalling pathway on osteoblast differentiation still needs to be thoroughly summarized and analysed, besides, the conclusions reached by some studies are also contradictory. Therefore, this review summarizes, analyses and discusses the recent studies on the role of Ca2+/CaN/NFAT signalling pathway in osteoblast biology, and summarized the different effects of a variety of compounds that have a regulatory effect on the Ca2+/CaN/NFAT signalling pathway in osteogenic differentiation.Many signal cascades in cells are activated by the increase of Ca2Osteoblasts are mainly derived from bone marrow mesenchymal stromal cells (BMSCs). BMSCs have the ability to differentiate into osteoblasts, adipocytes and chondrocytes, many transcription factors participate in the process of inducing BMSCs to differentiate into osteoblasts, such as runt‐related transcription factor 2 (RUNX2), β‐catenin and osteoblast‐specific transcription factor (Osterix), etc.2+/CaN/NFAT pathway has been shown to be activated by the non‐canonical Wnt signalling pathway during the differentiation of BMSCs into osteoblasts.2+ from ER into the cytoplasm by activating the IP3 receptor, and activates CaN by activating CaM to promote the nuclear translocation of NFAT.2+ concentration, thereby producing IP3, promoting the release of Ca2+ from ER and causing the increase in intracellular Ca2+ concentration.2+ in ER. When Ca2+ in ER is depleted, STIM1 aggregates and interact with the Orai1 protein on the cell membrane to open the store‐operated Ca2+ (SOC) channel and accelerate Ca2+ influx,2+/CaN/NFAT signalling pathway involved in.The differentiation of BMSCs into osteoblasts is regulated by a variety of signalling pathways, such as wingless‐type MMTV integration site (Wnt), transforming growth factor‐β/bone morphogenetic protein (TGF‐β/BMP), Hedgehogs and fibroblasts growth factor (FGF) signalling pathways.33.12+ can activate CaN by interacting with calmodulin (CaM).2+ binding sequences, and after binding Ca2+, a hydrophobic surface is exposed.2+ binding ability, CnA contains multiple domains, the more important of which are the phosphatase domain , CnB binding domain, CaM binding domain and the self‐inhibitory domain. The CaM binding domain can combine with the hydrophobic surface of CaM, and then be regulated by CaM.2+ binding CnB and CaM/CaM binding CnA, the conformation of CaN alters, the inhibitory zone separates itself from the phosphatase domain, causing CaN to be activated.2+/CaN/NFAT signalling pathway.Increased intracellular concentration of Ca3.22+ participates in a variety of signal transduction processes in cells. It can act as a secondary messenger or as a result of ion channel activation, affecting a variety of cell activities.2+ released during bone resorption will increase the local concentration of extracellular Ca2+, which functions as a coupling factor between osteoclasts and osteoblasts to chemoattractant the migration of osteoblasts through CaSR.2+ plays an important role in the process of osteogenic differentiation. During dentinogenesis, it involves the influx of extracellular Ca2+ and the release of Ca2+ from the intracellular Ca2+ storage.Ca2+ channel (L‐VGCC) participates in the proliferation of human BMSCs, MC3T3‐E1 osteoblasts and human periodontal ligament cells (hPDLCs), and mediates extracellular Ca2+ induced BMP‐2 signalling pathway activation and mineralization.2+, and the inhibitor of L‐VGCC nifedipine inhibits its osteogenic differentiation.L‐type voltage gated Ca+/Ca2+ exchanger (NCX) mediates Ca2+ flowing into the cells mainly through reverse exchange,2+ in osteoblasts and promote bone matrix mineralization.The Na2+ concentration and instantaneously mobilize intracellular Ca2+ flux.2+ influx caused by mechanically sensitive channels also promotes osteogenic differentiationThe CaSR belonging to the G protein‐coupled receptors can sense the extracellular Ca2+ channel of ER, can increase the intracellular Ca2+ concentration to promote osteogenic differentiation,2+ in the ER induced by IP3 causes STIM1 to accumulate at the junction of the ER and cell membrane, STIM1 interacts with Orai1 protein and activates the SOC channel, causing Ca2+ influx,Overexpression of Pannexin 3, which is the Ca2+ regulating osteogenic differentiation, there are also crosstalks between various Ca2+ channels. For example, Ca2+ can affect the proliferation and differentiation of osteoblasts through the mutual adjustment between CaSR‐L‐VGCC and SOC channel.In addition, in the process of Ca2+ (1.8–7.8 mM) in the culture medium of BMSCs, the cells exhibited larger area and circumference, as well as enhanced proliferation ability.2+ stimulation can cause the activation of downstream MAPK signalling pathways through Ca2+ influx, thereby promoting the expression of osteogenic differentiation‐related genes such as FGF‐2, BMP‐2, OPN, OCN and RUNX2.2+ did not affect the proliferation of DPCs, and did increase the mRNA levels of OCN and OPN, and enhanced their mineralization ability, but the mRNA levels of ALP and COL1A2 in cells decreased, ALP activity was also inhibited.2+ concentration increases to 50 mM, it will hinder the normal adhesion of cells.2+ treatment can enhance osteogenic differentiation and mineralization, but excessively high concentrations of Ca2+ may disrupt Ca2+ homeostasis and cause abnormal cell function.When supplemented with Ca3.32+ and activates a variety of downstream target proteins after binding Ca2+. It is precisely because there are many types of CaM downstream target proteins, the cell functions that CaM participates in are also diverse, such as inflammation, metabolism, apoptosis and so on.2+ flux by regulating Ca2+ channels, such as through IP3R and P/Q type calcium channel.2+ signals,Calmodulin is regulated by intracellular Ca3.42+ concentration.The calcium‐sensitive protein CaM can activate calcineurin under the condition of low and continuously increasing intracellular Ca3.52+ activates CaN through CaM, CaN dephosphorylates NFAT and exposes the nuclear localization sequence to cause its nuclear translocation.2+/CaN/NFAT pathway, CaN not only regulates the dephosphorylation and nuclear translocation of NFAT but also enhances its expression; therefore, NFAT as a transcription factor can cause its own self‐amplification effect.In the inactive state, NFAT protein localizes in the cytoplasm due to the hyperphosphorylation of its N‐terminal regulatory domain. After CaBesides, the promoter of the Fra‐2 gene contains three potential NFAT consensus sequences, and the combination of NFAT with Fra‐2 will cause the negative regulation of Fra‐2,42+/CaN/NFAT signalling pathway has been shown to be closely related with the physiological activities of osteoblasts. We have summarized compounds that have a regulatory effect on this signalling pathway and at the same time modulate osteoblastogenesis, aiming to provide new ideas for the exploration of treatment options for osteogenesis‐related diseases. Table 2+/CaN/NFAT signalling pathway on the differentiation of osteoblasts.Decreased differentiation or dysfunction of osteoblasts will lead to a variety of skeletal diseases. The Ca4.12+ signal in BMSCs, MC3T3‐E1 cells or osteoblasts, but Liou et al.2+ oscillation and Ca2+ signal activation.Xanthine derivative KMUP‐1 (7‐[2‐[4‐(2‐chlorophenyl)piperazinyl]ethyl]‐1,3‐dimethylxanthine) can inhibit phosphodiesterase (PDE) activity, Liou et al. found that 5–10 μM KMUP‐1 can induce osteogenic differentiation of BMSCs and MC3T3‐E1 cells and promote mineralization.4.22+ in osteoblast‐related cells has not been exhibited, but it has been demonstrated that 10–30 μM zinc inhibited the increase in Ca2+ concentration in BMMs induced by RANKL, and 30–100 μm zinc inhibited the CaN activity of BMMs.Zinc is essential in the process of skeletal development, 1–50 μM zinc has been shown to inhibit osteoblast apoptosis and promote the proliferation and differentiation of osteoblasts,4.32+ channel (VDCC) to promote Ca2+ influx, thereby increasing the intracellular Ca2+ concentration, the intracellular Ca2+ level increased the highest level when treated with 100 μM cyanidin.2+ influx,2+ concentration.2+ level caused by neurotensin,2+ concentration of RAW264.7 cells induced by RANKL.Cyanidin found in fruits and vegetables is a natural anthocyanin. Some previous studies have found that 50–200 μM cyanidin accelerated the proliferation, osteogenic differentiation and mineralization of MC3T3‐E1 cells.4.42+ oscillations and reduces Ca2+ level.Harpagoside is an iridoid glycoside extracted from harpagophytum procumbens var. sublobatum. Harpagide at concentrations of 0.032–4 μM promotes the osteogenic differentiation and mineralization of MC3T3‐E1 cells in a concentration‐dependent manner.4.52+ level induced by LPS in RAW264.7 cells, and also reduced the protein expression of the catalytic subunit of CaN. However, it is also reported that in airway smooth muscle cells (ASMCs), 1.5 and 2.0 mM ART significantly increased the intracellular Ca2+ concentration,2+ level of erythrocytes, Wu et al.2+ rapidly increased intracellular Ca2+ concentration under the treatment of 16–32 μM ART.Artesunate (ART) is a derivative of artemisinin, which has anti‐viral, anti‐tumour and anti‐malaria functions. Zeng et al.4.62+ influx by blocking Ca2+ channels except the two pore channel 2 (TPC2) and inositol 1,4,5‐triphosphate receptor 1 (IP3R1), causing reduction of intracellular Ca2+ concentration.The inhibitor of NADPH oxidase, apocynin, is a kind of methoxy‐substituted catechol. When MC3T3‐E1 cells are exposed to antimycin A and resulting in excessive ROS production, 0.01–1 μM apocynin can scavenge ROS, protect MC3T3‐E1 cells and promote their osteogenic differentiation.4.72+ levels and activate related downstream signals.2+ levels and activated the Ca2+ signalling pathway during the process of inducing osteoclast differentiation and bone resorption in BMMs.Alzheimer's disease is characterized by the loss of synapses and neurons in the elderly, and the accumulation of amyloid β peptide (Aβ) is its hallmark. Research by Yang et al.4.82+ binds to CaM, CaM activates a variety of target proteins, including calmodulin‐dependent protein kinase (CaMK). Choi et al. demonstrated that CaMKII participated in osteogenic differentiation of C2C12 mouse pre‐myoblast cell line induced by BMP‐4, and in the process of osteoblastogenesis, KN‐93 (2 mM), the inhibitor of CaMKII, blocked the osteogenic differentiation process of C2C12 cells induced by BMP‐4.In osteoblast precursor cells, after Ca4.9Trifluoperazine (TFP) can inhibit the activity of CaM and further restrain the activation of CaMKII. 10 μM TFP inhibits osteogenic differentiation, and also shows the ability to reduce the formation and mineralization of osteoblasts in the calvarial model of mouse pups.4.10Cyclosporin A and FK506 are CaN inhibitors and are widely used to reduce rejection reaction after organ transplantation. Low concentrations of CsA (less than 1 μM in vitro and 35.5 nM in vivo) have been shown to increase the expression of Fra‐2 to promote the transcription of osteogenic genes, thereby promoting osteogenic differentiation.52+/CaN/NFAT signalling pathway, which is an extremely important part of growth and development, is inextricably linked to bone formation. The regulation of each components in the signal pathway, such as activation, inhibition, overexpression, silencing, etc., often resulting in changes in the process of osteoblast differentiation in vivo and in vitro. In the process of osteoblast differentiation, the non‐canonical Wnt pathway triggers the activation of Ca2+/CaN/NFAT signalling pathway, then NFAT and Osterix form transcription complexes to induce the expression of downstream osteogenic‐related genes. Some research teams studied the effect of CaN on osteogenic differentiation by means of gene deletion or gain‐of‐function but came to diametrically opposite conclusions. The most important differences in the experimental methods of these teams lie in the different CaN subunits they target, and whether the intervention is limited to osteoblasts. In addition, the osteogenic function of NFAT has also been questioned, some studies claimed that it acted as a transcription factor to promote the expression of osteoblast‐related genes, whilst some other studies believed that NFAT inhibited the differentiation of osteoblasts by inhibiting Fra‐2. Most researchers agreed that low‐concentration CaN inhibitors promoted osteogenic differentiation, and high‐concentration CaN inhibitors suppressed the process of osteogenic differentiation, but no specific limit of the concentration of CaN inhibitor and the actual mechanism were given to explain the reason. In connection with the study of CaN inhibitors in osteoclasts, we speculate that immunophilin should also be included in the analysis of its influence on osteogenic differentiation.A variety of existing evidences indicate that the Ca2+/CaN/NFAT signalling pathway. However, we have found that some compounds positively regulate Ca2+ signal and promote osteogenic differentiation, whilst some compounds negatively regulate Ca2+ signal and promotes osteogenic differentiation as well. The reason for this paradox may lie in the different types of cells used in these studies for Ca2+ signal and the ability to regulate osteogenic differentiation, and the different application concentrations of the compounds, or the signalling pathway involved in the compounds driving osteogenic differentiation is not Ca2+/CaN/NFAT but other signalling pathways. The information we have collected and summarized can be used to investigate the relationship between the Ca2+/CaN/NFAT signalling pathway and osteogenic differentiation as well as providing some ideas for exploring better treatment options for regulating bone formation‐related diseases, and these remaining uncertain mechanisms require further research.A variety of existing compounds have the ability to promote or inhibit osteogenic differentiation, whilst regulating the Ca2+/CaN/NFAT signalling pathway and bone metabolism has been explored in many ways, this signalling pathway has a wide range of effects on cell fate, and the mechanisms involved are far‐reaching. There are still many unknown or unexplained relationships between Ca2+/CaN/NFAT signalling pathway and osteoblastogenesis, further exploration in this field is needed to broaden the way for the study of bone formation regulation and bone‐related diseases development.Over the years, the relationship between CaThe authors declare that they have no conflict of interest.W.X. and R.R. conceived the aims and structure of the review. R.R. and J.G. collected the articles and wrote the original draft of the manuscript. Y.C., Y.Z. and L.C. reviewed and edited the manuscript. W.X. acquired the funding. All authors have read and agreed to the published version of the manuscript.


Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing. The field of genome editing has grown rapidly as a result of the harnessing of programmable nucleases such as zinc-finger nucleases (ZFNs) , transcrDespite the high fidelity, HDR-based gene editing has limitations; it is restricted to the S and G2 phases of the cell cycle  and therIn this study, to measure NHEJ accuracy after the generation of the DSB by Cas9 nucleases, we used an exogenous DNA oligonucleotide inserted into the DSB site to prevent repetitive cleavage of the target site by the Cas9 nucleases. By inspecting the jointed DNA sequences between the exogenous DNA oligonucleotide and genomic DNA, we could determine whether the DSB was repaired flawlessly or not. Based on the measurements, we calculated NHEJ accuracy, respectively, at the DSB end proximal to a protospacer adjacent motif (PAM) and the DSB end distal to the PAM. As a result, we found that NHEJ accuracy is associated with their target sequences and is asymmetric between the DSB end proximal and distal to the PAM. In addition, we determined that the fraction of the insertion mutation of total mutations is primarily relevant to the erroneous repair of the NHEJ-mediated DSB repair.Cas9 nucleases can repeatedly cleave the target site until it is disrupted by mutations involving insertion, deletion or substitution, which might result in underestimating the degree of NHEJ accuracy. Inversely, the target site that has not been cleaved by Cas9 nucleases can be regarded as an accurate repair, which might lead to overestimating the degree of NHEJ accuracy. Thus, the evaluation of NHEJ accuracy after the generation of the DSB by Cas9 nucleases in intact cells is not easy. Modifying the target sequence after cleavage by the insertion of exogenous double-stranded oligodeoxynucleotides (dsODNs) into the DSB site is a simple way to mark the cleaved target DNA and restrict the number of cleavages by a Cas9 nuclease to one time, as in a previous study of genome-wide unbiased identifications of DSBs evaluated by sequencing (GUIDE-seq) . To thisHER4, EphA1, EphB4 and EMX1) to induce DSBs using Cas9 nucleases  in four genes  NHEJ accuracy is more related to the sequences flanking the DSB site rather than the gene type or the location of the DSB site and (ii) that the NHEJ-mediated DSB repair tends to be more reliable at the PAM-distal ends than at the PAM-proximal ends in general.We then investigated whether the above findings were conserved across cell types. To this end, an additional 18 sgRNAs targeting different genes for DSB generation were selected  and testIndel mutations are frequently generated at the DSB site during the NHEJ-mediated repair process. In addition, a previous study reported that individual targets of the CRIPSR-Cas9 have distinct preferences for the type of mutation . For exaHER4 gene of HeLa cells , between two PAM-proximal ends  or between one PAM-distal end and one PAM-proximal end  during the DSB repair A. Based La cells B.In the current study, we measured and compared the accuracy of the repair by the NHEJ pathway on the DSB generated by the Cas9 nucleases in human-originated cells. To assess the accuracy of the NHEJ-mediated repair for one working event, we added an exogenous DNA oligonucleotide that could label the DSB event of the cleaved target sites and prevent repetitive DSB generation by a Cas9 nuclease. From our strategy, we observed three major findings as follows: (i) NHEJ accuracy was independent of the target location or the cell types but dependent on the sequence flanking the DSB site (the average NHEJ accuracy was 75% in maximum), (ii) the NHEJ-mediated DSB repair was typically more error-prone at the PAM-proximal end of the DSB and (iii) the fraction of insertions in the total mutations showed a negative correlation with the degree of NHEJ accuracy.Our results suggested that two ends of the DSB site might go through differently via CRISPR-Cas9 nucleases. The release of cleaved DNA products by Cas nucleases is thought to be extremely slow ,24,25. POn the other hand, other molecules other than Cas nucleases may affect the accuracy of the DSB repair by the NHEJ pathway. It has been reported that the interaction between the RNA polymerase II and proteins associated with a DSB repair such as the catalytic subunit of DNA protein kinase (DNAPKcs) and Ku protein ,30,31. TAlthough we found a tendency that NHEJ accuracy was higher at the PAM-distal end compared with the PAM-proximal end, a few targets showed opposite results. Considering that these opposite tendencies in a few targets were conserved in both HeLa and HEK 293T cells, the results suggested that at least it was not due to the experimental variation. One possible explanation for this inconsistency is that the sequences flanking the DSB site might be important in determining NHEJ accuracy at the PAM-distal end and the PAM-proximal end. If the sequence at the PAM-distal end is vulnerable to the erroneous repair, the degree of NHEJ accuracy at the PAM-distal end could be lower than that at the PAM-proximal end. Another interesting finding of our study was that there was an inverse correlation between the fraction of insertions of the mutations and the degree of NHEJ accuracy at the PAM-proximal end of the DSB. This result indicated that an inaccurate repair was more frequent at the PAM-proximal end of the DSB and that it might be associated with previous observations that Cas9 nucleases frequently generate staggered ends with 5′ overhang at the cleavage site resulting in a 1 bp insertion  mutation more abundantly ,36,37,38The higher NHEJ accuracy at the PAM-distal end of the DSB site suggested the feasibility of precise DNA deletion by the generation of two DSBs. Indeed, we confirmed a much higher NHEJ accuracy in the PAM-in configuration . Many kiIn summary, our current study investigated the features of a DSB repair mediated by the NEHJ pathway and will be informative in its application in diverse ways including gene therapy.HeLa  and HEK 293T  cell lines were maintained in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 unit/mL penicillin and 0.1 mM non-essential amino acids.All sgRNAs were designed using a Cas-designer to target each gene or site and included a 5′-NGG-3′ PAM sequence . A pRG2-5 cells according to the manufacturer’s protocol. SE solution  and SF solution  were used for HeLa cells and HEK 293T cells, respectively. In the presence of the dsODN, the SpCas9 plasmid (500 ng), the sgRNA plasmid (500 ng) and the dsODN  were electroporated into 1.0 × 105 cells using the same protocol. For the large deletion experiment, the SpCas9 plasmid (750 ng) and each sgRNA plasmid (250 ng) were transfected. The genomic DNA was isolated from the whole cell population using NucleoSpin Tissue  72 h after transfection. A Nucleoflector 4D  was used for HeLa cells and HEK 293T cells. The SpCas9 plasmid (500 ng) and the sgRNA plasmid (500 ng) were electroporated into 1.0 × 10https://github.com/brwnj/fastq-join [http://www.rgenome.net/cas-analyzer/) [The targeted sites were amplified with a Phusion HF DNA polymerase  to produce a sequencing library. The libraries were sequenced using MiniSeq with the TruSeq HT Dual Index system and a paired-end system . The NGS results were joined using a Fastq-join tool  . https://github.com/Gue-ho/NHEJ_accuracy).The analysis program was developed using Python 3. The analysis of the accuracy of the NHEJ pathway was performed through the following steps: (i) The Fastq-join sequence was sorted by the 12 bp indicator sequence that was 70 bp apart from the cleavage at both the PAM-distal end and the PAM-proximal end. (ii) The same read was counted and removed when the count of read was 1. (iii) If the reads had the WT marker that was ±5 bp at the cleavage sites of the reference sequence, the reads were classified as a “wild type”, which was excluded from the calculation of the repair accuracy. (iv) If the reads had 10 bp in the inserted dsODN sequence, the reads were classified as “dsODN inserted reads”. (v) The remaining reads were classified as “mutation reads”. (vi) In the dsODN inserted reads, if the reads had accurately 10 bp at the end of the dsODN and at the end of the cleavage sites, the reads were classified as an “accurate repair”. The analysis process for the accuracy of dual gRNAs was the same as (i)–(iii) of the NHEJ accuracy analysis. The accuracy was determined by the marker that was 5 bp before and after the two target cleavage sites. The script used for the analysis is available in github . A parametric statistical test (paired t-test and a one-way ANOVA) was used only for the data set that passed the Shapiro–Wilk normality test. Otherwise, the Wilcoxon signed rank test and Kruskal–Wallis test were used.Statistical tests were described in the main text. All statistical tests were two-sided (*


Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.Regulation of IP Calcium signals initiated by IP3 receptors in ER membranes regulate most cellular activities. Here, the authors show that KRas-induced actininteracting protein (KRAP) tethers a small subset of IP3 receptors to actin and licenses them to evoke cytosolic calcium signals. Most of these signals are due to the opening of Ca2+-permeable channels, which then allow Ca2+ to flow into the cytosol across either the plasma membrane (PM) or the membranes of intracellular organelles, primarily the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate receptors (IP3Rs), the most widely expressed of these Ca2+ channels, reside in the membranes of the ER from which they release Ca2+ when they bind IP33. This Ca2+ flux delivers Ca2+ to the cytosol and to the cytosolic surface of other organelles, notably mitochondria4 and lysosomes5. By depleting the ER of Ca2+, IP3-evoked Ca2+ release also stimulates store-operated Ca2+ entry (SOCE) across the PM. Stromal interaction molecule 1 (STIM1), which straddles the ER membrane, detects the loss of ER Ca2+ through its luminal Ca2+-binding sites causing it to unfurl cytosolic domains. These domains reach across narrow membrane contact sites (MCS) between the ER and PM to contact Orai Ca2+ channels, causing them to open and allow Ca2+ to flow into the cell through the SOCE pathway7. IP3Rs are therefore Ca2+-signalling hubs: in all animal cells, they link the extracellular stimuli that evoke the formation of IP3 to delivery of Ca2+ from the ER to the cytosol or other organelles, and to the regulation of Ca2+ entry across the PM through SOCE.Cytosolic Ca3 is not alone sufficient to stimulate the opening of an IP3R. Instead, IP3 binding to all four subunits of a tetrameric IP3R8 primes it to respond to Ca2+, which then evokes channel opening3. In the presence of IP3, IP3Rs can thereby evoke regenerative signals through Ca2+-induced Ca2+ release (CICR). The smallest of these regenerative events are Ca2+ puffs, which are local cytosolic Ca2+ signals that arise from coordinated openings of a few clustered IP3Rs as Ca2+ released by an open IP3R stimulates the opening of its neighbours9 . We show that all Ca2+ signals, whether local or global, require KRAP-mediated licensing of IP3Rs and that endogenous KRAP limits the capacity of IP3Rs to respond. This additional, and obligatory, level of IP3R regulation reveals an important role for the actin cytoskeleton in regulating IP3-evoked Ca2+ signals and suggests mechanisms whereby local depletion of the ER might control SOCE.Here, we identify this additional form of regulation and demonstrate that IP3Rs tagged with enhanced green fluorescent protein (EGFP) to address the mechanisms that license immobile IP3Rs adjacent to the PM to evoke Ca2+ puffs20. IP3R1 is the major subtype in HeLa cells22, but we showed previously that an antibody to EGFP immunoprecipitated all three IP3R subtypes from EGFP-IP3R1 HeLa cells in the same ratio as their overall expression, confirming that EGFP-IP3R1 subunits assemble with the other IP3R subtypes20. Thus, EGFP-IP3R1 puncta probably report the presence of all three IP3R subtypes in EGFP-IP3R1 HeLa cells.We used HeLa cells with endogenous type 1 IP2+ leaking across the PM might provide a local increase in cytosolic free Ca2+ concentration ([Ca2+]c) that then selectively stimulates IP3Rs immediately beneath the PM. Previous work with SH-SY5Y neuroblastoma cells9 and human embryonic kidney (HEK) cells11 established that Ca2+ puffs do not require extracellular Ca2+. We confirmed these findings in HeLa cells by demonstrating that the frequency and amplitude of Ca2+ puffs evoked by photolysis of caged IP3 (ci-IP3) were unaffected by removing extracellular Ca2+  revealed that 27 ± 7% of EGFP-IP3R1 clusters colocalize with actin filaments  reported previously20. Live-cell imaging confirmed that immobile IP3R puncta selectively associate with actin , was originally identified as a large actin-binding protein that is over-expressed in a colon cancer cell line expressing activated KRas33. KRAP is now known to be widely expressed34 and to associate with all three IP3R subtypes36. Results from co-immunoprecipitation and immunocytochemical analyses suggest that the N-terminal region of KRAP may interact with IP3Rs, while the C-terminal may interact with actin , now designated IP35  and TIRFM, we showed that IP3Rs and KRAP cluster around the same centroid in each punctum. However, IP3Rs were often quite widely distributed within a punctum, whereas KRAP was more tightly clustered 46 together with siRNA to reduce expression of all IP3R subtypes to examine any effects of IP3Rs on the subcellular distribution of KRAP and STIM1. Loss of Ca2+ from the ER caused STIM1 puncta to accumulate beneath the PM, and these puncta accumulated alongside, but not coincident with, KRAP  or additional IP3Rs . Indeed, recent evidence suggests that global Ca2+ signals may not be entirely associated with underlying Ca2+ puffs12.Caays Fig. . Since a3R mobility and Ca2+ signals after photolysis of ci-IP3, and then fixing cells to visualize KRAP, we established that Ca2+ puffs occur at sites populated by KRAP and immobile IP3R puncta  or global, require licensing of IP3Rs by KRAP 53, Bcl2 (B-cell lymphoma 2)54, annexin A155 and many other signals inhibit IP3Rs56, and they may be lost when cells are disrupted, perhaps thereby alleviating the need for KRAP in analyses of broken cells.How can an obligatory need for KRAP in intact cells be reconciled with evidence that IP3-evoked Ca2+ signals in all cells arise immediately beneath the PM. We consider two possible explanations. It may be that in other cells or in response to appropriate regulatory signals, IP3Rs may be licensed by association with KRAP in different subcellular locations, allowing them to initiate Ca2+ signals away from the PM. Alternatively, and consistent with recent analyses of local and global Ca2+ signals in HEK cells12, a flurry of Ca2+ puffs mediated by licensed IP3Rs may provide a signal 12 that ignites the activity of unlicensed IP3Rs. Hence, and consistent with our observations  to evoke global Ca2+ signals. The second scheme also aligns with evidence from pancreatic acinar cells. In these cells, KRAP is located at the apical pole of each cell57, where IP3-evoked Ca2+ signals initiate before propagating via IP3Rs and ryanodine receptors to the basal pole;58 disruption of the actin cytoskeleton perturbs these local Ca2+ signals and the apical distribution of IP3Rs26.A second issue arises from observations that not all IP3Rs by KRAP is likely to have further implications. By positioning responsive IP3R clusters alongside the MCS where SOCE occurs, KRAP may ensure that activation of IP3Rs selectively depletes the ER that is best placed to regulate SOCE. We speculate that this may allow substantial local depletion of small ER compartments close to the PM and thereby allow local digital regulation of SOCE , is both the source of IP3 and a key regulator of actin filaments62, suggesting that PIP2 may control both licensing of IP3Rs and their activation by IP3 benzyl-myo-inositol 1,4,5-trisphosphate hexakis(propionoxymethyl) ester) was from SiChem . Acti-stain 670 phalloidin (# PHDN1-A) was from Cytoskeleton . BAPTA ethane-N,N,N′,N′-tetraacetic acid) was from Molekula . Histamine was from Sigma-Aldrich. Bovine serum albumin (BSA) was from Europa Bio-Products . Withaferin A was from AdipoGen Life Sciences . AlexaFluor-568 phalloidin and restriction enzymes were from ThermoFisher. Human fibronectin was from Merck Millipore . Plasmids encoding the following proteins were from: LifeAct-mCherry-N1 (Addgene #40908)63, LifAct-7-iRFP670 (Addgene #103032)64, human KRAP with an N-terminal Myc-DDK tag , mCherry-keratin (Addgene #55066) and mCherry-vimentin-N-18 . Cytochalasin D and latrunculin-A were from Tocris . Antibodies  were from: β-actin ; KRAP , the same KRAP primary antibody was custom-conjugated to YF-594 for TIRF imaging during STORM analyses of IP3Rs ; GFP Tag-AlexaFluor-647 ; IP3R1 ; IP3R2 ;65 IP3R3 ; STIM1 ; vimentin ; GFP-Booster Atto488 ; donkey anti-rabbit IgG-HRP ; donkey anti-mouse IgG-HRP ; goat anti-rat IgG-HRP ; goat anti-rabbit AlexaFluor-594 ; goat anti-rabbit AlexaFluor-647 ; goat anti-mouse AlexaFluor-568 ; and goat anti-chicken AlexaFluor-568 . Additional sources of materials are provided in relevant methods.Cal-590 AM, Calbryte 590 AM and Fluo-8 AM were from AAT Bioquest . A membrane-permeant form of caged-IP3Rs 8, HeLa cells, STIM1-EGFP HeLa cells66 and EGFP-IP3R1 HeLa cells20 were cultured in Dulbecco’s modified Eagle’s medium/F-12 with Gluta-MAX  supplemented with foetal bovine serum . The cells were maintained at 37 °C in humidified air with 5% CO2 and passaged every 3-4 days using Gibco TrypLE Express (ThermoFisher). We have reported an extensive characterization of the EGFP-IP3R1 HeLa cell line, in which monomeric EGFP was attached to the N-terminal of all copies of the endogenous IP3R1 gene (ITPR1) using TALENs (transcription activator-like effector nucleases)20. The characterization included evidence that EGFP-IP3R1 is functional. In the STIM1-EGFP HeLa cell line, CRISPR/Cas9 was used to add monomeric EGFP to the C-terminal of one of the two STIM1 genes66. For STIM1-EGFP HeLa cells, we have confirmed that the only fluorescent protein in the cells is STIM1-EGFP and that histamine-evoked Ca2+ signals and SOCE are unperturbed by the gene-editing46. KRAP is up-regulated in some cells with mutant KRas33, but we confirmed by pyrosequencing  that there were no mutations in codons 12, 13 (exon 2), 59, 61 (exon 3), 117 or 146 (exon 4) of the KRAS gene in the EGFP-IP3R1 HeLa cells.HEK cells, HEK cells devoid of all IP-1). Regular screening confirmed that all cells were free of mycoplasma. The authenticity of the EGFP-IP3R1 HeLa cells  and HEK-3KO cells  was verified by short-tandem repeat profiling.For imaging, cells were grown on 35-mm glass-bottomed dishes  coated with human fibronectin (10 μg ml3R1 HeLa cells grown on Cellvis imaging dishes were transfected with the appropriate plasmid (1–2 µg DNA per dish) using ViaFect  transfection reagent (3 µl per 1 µg DNA) according to the manufacturer’s instructions. Cells were used after 24 h.For transient transfections, EGFP-IP2 culture flasks were harvested using either enzyme-free cell dissociation buffer (ThermoFisher) or, for siRNA experiments, by scraping cells into lysis medium  containing a protease inhibitor mini-tablet with EDTA . Cells were then lysed by incubation in LM at 4 °C for 1 h, sonicated , and the supernatant was collected . Since actin-associated proteins are more resistant to detergent-extraction, and KRAP facilitates IP3R association with actin  analyses of IP3Rs before and after KRAP knock-down  and transferred onto an iBlot PVDF membrane using an iBlot gel-transfer device (ThermoFisher). The PVDF membrane was blocked by incubation  in TBST, which comprised Tris-buffered saline  supplemented with bovine serum albumin  and Tween-20 (0.1%). The blocked membrane was incubated  with primary antibody (in TBST with 1% BSA), washed (3 × 5 min) in TBST, and incubated  with horseradish peroxidase (HRP)-conjugated secondary antibody in TBST with 1% BSA. The antibodies used and their dilutions are listed in the Materials section. After further washes with TBST (3 × 5 min), HRP was detected using ECL Prime Western blotting detection reagents  and a PXi chemiluminescence detection system . Where main figures show only part of a WB, the complete WB is shown in Supplementary Fig. 2+ signals  were transfected with siRNA directed against KRAP  or a non-silencing (NS) control siRNA  using either siPORT NeoFX transfection reagent  or a Neon transfection system (ThermoFisher) according to the manufacturer’s instructions. The two KRAP siRNAs were used interchangeably and with indistinguishable knockdown efficiencies  or NS siRNA . Cells were used after 72 h.For siRNA-mediated knockdown of the three IPGAAGTGTTCAGACATGA in the mutated form (mutated residues are underlined). The modified and native DNAs encode the same protein sequence.A siRNA-resistant version of the KRAP plasmid was prepared by digesting the plasmid encoding human KRAP  at Bst1107I and ScaI restriction sites. This was followed by the introduction of a DNA string of the excised region containing silent mutations at the siRNA target site with 30 bp flanking regions of the two restriction sites (ThermoFisher), into the plasmid using Gibson assembly . The sequence of the DNA from which the siRNA-targeted mRNA sequence derives (bp 2011–2029 of the coding sequence) was: GCTAAATGCAGTGATATGA in the native DNA, and GCCells grown on fibronectin-coated Cellvis glass-bottomed dishes were washed three times in phosphate-buffered saline , fixed with paraformaldehyde (4%) in PBS (30 min), washed three-times in PBS, and permeabilized by incubation (5 min) in PBS containing Triton X-100 (0.25%). After three washes with PBS, cells were incubated (1 h) in PBS containing BSA (5%), washed briefly, and then incubated (1 h) with primary antibody  in PBS containing BSA (3%). After three washes with PBS, cells were incubated (1 h) with an appropriate AlexaFluor-conjugated secondary antibody, fluorescent phalloidin or GFP-Booster  in PBS containing BSA (3%). The cells were then washed three times with PBS before imaging. For matched analyses of live and immunostained cells Fig. , fixatioFluorescence microscopy used an inverted Olympus IX83 microscope equipped with a 100× oil-immersion TIRF objective , a multi-line laser bank  and an iLas2 targeted laser illumination system . In some experiments that required larger fields of view, we used a ×60 (NA 1.45) oil-immersion TIRF objective Fig. . Excitat67 using the Live-SR module and optical reassignment processing to give a lateral resolution of approximately 120 nm.Super-resolution confocal microscopy used an inverted Nikon Eclipse Ti2 microscope equipped with a ×100 oil-immersion TIRF objective (NA 1.49), an X-Light V3 spinning-disc confocal unit with 50-µm pinholes (CrestOptics), and a Live-SR super-resolution module . Excitation light  from a laser diode illuminator  was transmitted through a quad-band filter set (405/470/555/640 nm). Emitted light was passed through an appropriate emission filter  and detected with a Prime 95B camera. Super-resolution was achieved by multifocal structured illuminationhttps://fiji.sc)68, respectively. Confocal images were deconvolved using the Microvolution deconvolution algorithm (version 2015.05)69. All images are presented in RGB colour (16-bit) format.Before analysis, all fluorescence images were corrected for background by subtraction of fluorescence detected from a region outside the cell. Image capture and processing used MetaMorph Microscopy Automation and Image Analysis Software  and Fiji , permeabilized and immunostained. For identification of KRAP by STORM, we used a rabbit polyclonal primary antibody (1:400) and goat anti-rabbit AlexaFluor-647 secondary antibody (1:500); IP3Rs were identified in TIRF from their EGFP fluorescence. To identify IP3Rs using STORM, EGFP-IP3R1 was visualized using GFP Tag-AlexaFluor-647 (1:400); KRAP was identified in TIRF using the same primary antibody used for STORM analyses, but conjugated directly to YF-594 . The medium used for STORM comprised: Tris-HCl (50 mM), pH 8.0, NaCl (10 mM), glucose (555 mM), catalase , glucose oxidase , 2-mercaptoethanol  and cyclooctatetraene . For STORM imaging, stochastic blinking of individual fluorophores was achieved by first bleaching cells in semi-TIRF mode using full laser power (647 nm). Images  were then acquired in TIRF at a lower laser power (647 nm) using a TIRF objective  and an Andor iXon Ultra EMCCD camera. Image acquisition and fitting used WaveTracer (MetaMorph) to detect genuine blinking events followed by fitting a centroid to each blinking event within each 10 nm × 10 nm pixel. Point localizations of blinking events from each of the 20,000 frames were collated to construct a super-resolution STORM image20. STORM images generated using four-colour TetraSpeck microspheres  confirmed that there was no chromatic aberration or drift during image acquisition. The lateral resolution for STORM  determined using TetraSpeck microspheres was of 21.8 ± 4.0 nm, and 24.2 ± 5.6 nm when determined using STORM images generated with AlexFluor 647.The methods used to generate STORM images were exactly as described20. Briefly, time-lapse images (~1 frame per second) were acquired in epifluorescence mode using a 488-nm laser. A circular ROI (diameter = 2.3 μm) was rapidly photobleached by raster scanning at either a perinuclear or peripheral region of a cell, using a 395-nm laser and an iLas2 laser illumination system. Recovery of fluorescence was then recorded. Images were background-corrected by subtracting the fluorescence recorded from a region outside the cell. The mobile fraction was calculated using the ImageJ plugin, FRAP profiler (http://worms.zoology.wisc.edu/ImageJ/FRAP_Profiler.java).The methods used for wide-field FRAP measurements, after photobleaching a circular region of interest (ROI), were exactly as described previously3R); the need to compare two or three colocalized fluorophores; our use of TIRF and spinning-disc confocal microscopy, where the latter contributes more out-of-focus fluorescence; and our need to compare both punctate  and elongated  structures, where only the punctate structures are amenable to object-based analyses.These analyses required different methods to accommodate our need to quantify relationships between fluorophores where only subsets of particles were colocalized 44 to quantify their colocalization by measuring centre-to-centre distances between IP3R and KRAP puncta  are then calculated. Since these measurements provide sub-pixel localization accuracy, we considered IP3R and KRAP puncta to be colocalized if their centre-to-centre distance was <160 nm . Our aim was to use similar criteria for colocalization for each analysis, while ensuring that the number of pixels used to define colocalization for each imaging method came closest to the theoretical lateral resolution of the method. To assess the statistical significance of the colocalization, the algorithm randomizes (100 iterations) the segmented image of all KRAP puncta before re-calculating the centre-to-centre distances44. This analysis was used to provide the cumulative distribution of centre-to-centre distances for unperturbed IP3R puncta and randomly distributed KRAP puncta with its 95% confidence interval (CI)  and coordinates for EGFP-IP3R puncta derived from Fiji.Colocalization analyses of Ca3R or KRAP, actin images were first Gaussian-filtered (σ = 0.5) to remove uneven background fluorescence. We then used the Fiji JACoP plugin70 to calculate the Mander’s split coefficient, which reports the fraction of IP3Rs or KRAP colocalized with actin  with IPtin Fig. .3R and KRAP puncta in confocal sections, deconvolved images were Gaussian-filtered (σ = 0.5) to remove noise. For confocal sections remote from the coverslip, a peripheral region of interest (ROI) (i.e. close to the PM) was defined by an annulus extending 15 pixels (i.e. 2.4 μm) inward from the cell boundary, while the remaining core formed the central ROI  using the JACoP plugin70 for each ROI  was used for analysis. Similar methods were used for analyses of vimentin filaments. Deconvolved confocal images were Gaussian-filtered for actin staining (σ = 0.5). Centre-to-centre distances between IP3R and KRAP puncta were then measured using the DiAna plugin44 for ROI within and outside the actin mask. We considered IP3R and KRAP puncta to be colocalized if their centre-to-centre distance was <160 nm .Since methods for quantifying colocalization of more than two fluorophores by object-based colocalization methods are not well developed, we measured the colocalization of IPtin Fig. . For eac2+ puffs, EGFP-IP3R1 HeLa cells were grown on fibronectin-coated Cellvis dishes and transfected with siRNA (see the section on Treatment with siRNA) or plasmid encoding KRAP (see the section on Cell culture and transfection). Before use, cells were washed twice with HBS and incubated (1 h) in HBS containing Cal-590-AM  or Calbryte 590-AM  and ci-IP3/PM (1 μM), washed twice with HBS, and then incubated  in HBS containing EGTA-AM (5 μM). EGTA is a slow Ca2+-buffer that limits regenerative propagation of global Ca2+ signals without perturbing Ca2+ puffs71. After two further washes, cells were incubated in HBS (30 min 20 °C), washed and imaged in HBS at 20 °C. Fluorescence was recorded for 32.5 s by TIRFM  with illumination for 25 ms in every 50-ms capture interval to minimize photobleaching. A flash of ultraviolet (UV) light  was delivered after 2.5 s to photolyse ci-IP3. Images were collected using MetaMorph, corrected for background fluorescence, and Ca2+ puffs were detected and analysed using the FLIKA algorithm (version 1)72. Similar methods  were used for analyses of the effects of histamine on Ca2+ puffs and global increases in [Ca2+]c  or photolysis of ci-IP3   Fig. ; the res3R1 HeLa cells were grown in clear-bottomed 96-well plates  coated with fibronectin (10 μg ml−1) (see the section on Cell culture and transfection). Cells were then washed and loaded with Fluo-8 by incubation in HBS with Fluo-8 AM , washed and incubated in HBS  before experiments. A FlexStation 3 plate-reader (Molecular Devices) was used to measure Fluo-8 fluorescence at 1.4-s intervals at 20 °C . Responses to histamine were determined in HBS, and responses to ionomycin were determined after the addition of BAPTA in Ca2+-free HBS to chelate extracellular Ca2+ . Fluorescence (F) was recorded  and calibrated to [Ca2+]c from [Ca2+]c = KD(F − Fmin)/(Fmax − F), where the KD of Fluo-8 for Ca2+ is 389 nM, and Fmin and Fmax are the minimal and maximal fluorescence signals determined after addition of Triton X-100 (1%) with either BAPTA (2.5 mM) for Fmin or CaCl2 (10 mM) for Fmax.EGFP-IP3R puncta by overlaying pseudocoloured images collected at intervals of 30 s, such that the two colours (green and magenta) overlay for immobile IP3Rs 20. But for all quantitative analyses, we used single-particle tracking 73 and trajectory classification 74 to identify immobile IP3R puncta. We confirmed the congruence of the two approaches previously20. TrackMate requires selection of unique thresholds for detection of puncta in each image73, but we confirmed the validity of the thresholds by both manual inspection of images and by demonstrating that the fraction of whole-cell fluorescence attributed to puncta was consistent. For example, in TIRF analyses of cells treated with NS or KRAP siRNA . Using TrackMate, the sub-pixel localization of each IP3R punctum in time-lapse TIRF images  was obtained before linking the particles to generate track segments, followed by gap-closing to link the track segments. A displacement threshold of 1.5 μm s−1 was employed to track and link particles since the maximal velocity of the particles did not exceed this limit. TraJClassifier, a validated plugin for trajectory classification74, was used to classify single-particle trajectories from TrackMate. This algorithm uses a machine-learning approach to classify single-particle trajectories into diffusive, sub-diffusive, confined and directed trajectories. For inclusion in the classification analysis, the minimum trajectory length was 45 frames. This algorithm does not specifically identify immobile particles, which fall within the category of sub-diffusive particles. From each sub-diffusive trajectory, we used the exponent (α) from the plot of mean squared displacement  versus time (t) for anomalous sub-diffusion 75 and classified particles with α < 0.1 as immobile. The value (α < 0.1) was chosen from the analysis of simulated trajectories performed by Thorsten Wagner (University of Dortmund).For convenience, we often show immobile IPn independent analyses. Statistical comparisons used paired (where indicated) or unpaired, two-tailed Student’s t tests, or analysis of variance (ANOVA) with the Bonferroni’s correction for multiple comparisons . Significance levels are shown as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Variances of the product of two variables (for Supplementary Fig. 76 (details in Supplementary Table Most results are presented as mean ± s.d. or s.e.m. from Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Supplementary Video 7Supplementary Video 8Supplementary Video 9Supplementary Video 10Description of additional supplementary filesReporting Summary