Visual Abstract

Although the mechanism of NK cell activation is still unclear, the strict calcium dependence remains the hallmark for lytic granule secretion. A plethora of studies claiming that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis accompanied by weak target cell lysis has been published. However, there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity. In our study, we observed that small-molecule inhibitor UNC1999, which suppresses global H3K27 trimethylation (H3K27me3) of human NK cells, induced a PKD2-dependent calcium signal. Enhanced calcium entry led to unbalanced vesicle release, which resulted into fewer target cells acquiring lytic granules and subsequently being killed. Further analyses revealed that the ability of conjugate formation, lytic synapse formation, and granule polarization were normal in NK cells treated with UNC1999. Cumulatively, these data indicated that induced calcium signal exclusively enhances unbalanced degranulation that further inhibits their cytotoxic activity in human NK cells.

Immune evasion is a major obstacle to designing effective anticancer treatment strategies. The main mechanisms of immune evasion include alteration to the speed and efficacy of immune cells in killing cancer cells. The NK cell has the ability to nonspecifically recognize and kill cancer cells without presensitization, therefore playing an important role in immunosurveillance (1). The effector function of NK cells, characterized by the speed and effectiveness of NK cells killing target cells, is largely dependent on Ca2+ influx (2). Therefore, elucidating the correlation between Ca2+ influx level and NK cell cytotoxicity is necessary.

During target cell recognition, NK cells acquire activation signals and undergo a series of biological processes, including extracellular Ca2+ influx, cytoskeletal rearrangements, and immune synapse (IS) formation, followed by polarization of the granules toward the target cells. The end result in the lytic interaction is the exocytosis of lytic granules, which entails programming the target cell for lysis (35). In addition, the NK cells can detach from the target and kill again, a process known as serial killing (6). Results from previous studies have supported the association of Ca2+ influx with NK cell cytotoxicity. By analyzing NK cells from ORAI1-deficient patients, Bryceson et al. (7) observed that store-operated Ca2+ entry via ORAI1 is critical for target cell-induced lytic granule exocytosis in NK cells. Additionally, Schwindling and his colleague (8) reported that mitochondrial polarization to the IS is Ca2+ dependent. Lytic granule accumulation at the IS is a crucial step during the killing of the target cell by NK cells and has been reported to be indirectly modulated by the internal Ca2+ concentration (9).

Ca2+ release-activated Ca2+ channels, composed of ORAI1, form the dominant Ca2+ influx pathways in NK cells (10). ORAI1 are activated through direct physical interaction with STIM1, a sensor of Ca2+ depletion located in the membrane of the endoplasmic reticulum (ER) (11). Because the STIM1:ORAI1 protein ratio plays an important role in exocytosis modulation (one ORAI1 channel needs twelve STIM1 molecules to carry the maximum Ca2+ release-activated Ca2+ current) (12), shifting the expression of either STIM1 or ORAI1 decreases the Ca2+ influx, further resulting in decreased cytotoxicity of NK cells; so, how does one upregulate Ca2+ influx and what will happen when Ca2+ influx is upregulated in NK cells? In our study, we inadvertently noticed that small-molecule inhibitor UNC1999, which suppresses H3K27me3 modification of NK cells, also induced a PKD2-dependent calcium signal. Further analyses showed that enhanced Ca2+ influx leads to unbalanced vesicle release that, in turn, inhibits their cytotoxic activity. Our results demonstrate that optimized Ca2+ entry is extremely important for the balanced vesicle release of NK cells.

The NK92MI cell line was purchased from American Type Culture Collection. Cells were maintained in α-MEM (Life Technologies) supplemented with 2 mM l-glutamine (Life Technologies), 0.2 mM I-inositol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), 0.1 mM 2-ME (Invitrogen), 12.5% FBS (Life Technologies), and 12.5% horse serum (Life Technologies). K562 target cells (kindly provided by Tao Cheng of the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences) were cultured in RPMI 1640 (Life Technologies) supplemented with 1% penicillin/streptomycin (HyClone Laboratories) and 10% FBS (Life Technologies). Human peripheral blood NK cells were isolated by negative magnetic selection (Miltenyi Biotec) and cultured in RPMI 1640 (Life Technologies) supplemented with 1% penicillin/streptomycin (HyClone Laboratories), 10% FBS (Life Technologies) and 500 U/ml human rIL-2 (PeproTech). Prior to their use, UNC1999 (Selleck Chemicals) and GSK343 (Selleck Chemicals) were dissolved in DMSO to make 5 mM stock solutions. Blood samples were obtained from healthy donors who had signed informed consents in accordance with the Declaration of Helsinki.

As previously described (13), the degranulation assay for cells stained with fluorochrome-conjugated mAbs for CD56 (562794; BD Biosciences; 20-0564; Tonbo Biosciences,) and CD107a (555801; BD Biosciences; 328608; BioLegend) was done by flow cytometry (LSRFortessa; BD Biosciences).

Target cells were labeled with 1 μM CFSE (BioLegend) for 10 min at 37°C. After incubation, they were washed once using RPMI 1640 supplemented with 10% FBS. The NK cells and target cells were coincubated at E:T ratios of 10:1, 5:1, and 2.5:1. Basal cell death was measured using target cells that had been incubated alone. After 4 h of incubation (12 h for long-term killing), cell mixtures were washed twice using PBS, incubated for 20 min at room temperature in a buffer solution with 20 μg/ml 7-AAD (BioLegend), and analyzed by flow cytometry. NK cell–mediated cytotoxicity was assessed by determining the percentage of dead target cells (CFSE+/7-aminoactinomycin D+).

Real-time cytotoxicity was assessed using a Real-Time Cellular Analyzer (RTCA) system (ACEA Biosciences). Briefly, HeLa cells were washed and resuspended in RPMI 1640 supplemented with 10% FBS at a density of 5 × 104 cells/ml. Next, 100 μl of HeLa cells was added to each well of E-Plate 16. A microelectrode sensor array, which could identify biological information associated with physiological functions of the cells by detecting changes in impedance values, was mounted at the bottom of each cell growth well of E-Plate 16. E-Plate 16 was placed on the RTCA Station in the incubator. After 12 h, the plate was removed from the station and NK92MI cells were added to each well at E:T ratios of 10:1, 5:1, and 1:1. The plate was placed back on the RTCA Station and monitored in real time to observe the effects of NK92MI cells on HeLa cells.

NK cells (1 × 106) were stained with allophycocyanin-Cy7-conjugated CD56 mAb (318332; BioLegend,), whereas K562 cells (1 × 106) were labeled with 1 μM CFSE (BioLegend) for 10 min at 37°C. Cells were washed and resuspended in RPMI 1640 medium supplemented with 10% FBS at a density of 1 × 106 cells/ml. Next, 100 μl of NK cells was added to 100 μl of K562 cells, followed by centrifugation at 20 × g for 1 min. After removing 150 μl of the supernatant, cells were stimulated by incubation at 37°C for 5 or 15 min. Reactions were stopped by adding 300 μl of ice-cold paraformaldehyde (0.5%). Conjugates were detected by flow cytometry (LSRFortessa; BD Biosciences).

NK cells (1 × 106) and K562 target cells (1 × 106, prestained with CFSE for synapse analysis) were mixed and centrifuged at 30 × g for 3 min, followed by incubation for 20 min at 37°C. Cells were gently resuspended and treated with 100 μl fixation/permeabilization solution (BD Biosciences) for 20 min. They were then transferred to poly-d-lysine–coated slides (Sigma-Aldrich) for 15 min at 37°C. For synapse analysis, cells were stained with phalloidin (Invitrogen) and evaluated by laser scanning confocal microscopy. For polarization analysis, cells were stained with α-tubulin (Abcam) and perforin (BioLegend) and evaluated by laser scanning confocal microscopy.

NK92MI cells were fixed, permeabilized, stained with allophycocyanin-conjugated perforin mAb (308109; BioLegend) and PE-conjugated perforin mAb (561142; BD Biosciences), and analyzed using flow cytometry (BD Biosciences).

NK92MI cells were loaded with 5 μM Fluo-3 (Invitrogen) in FBS-free RPMI 1640 medium for 60 min at 37°C, washed twice, and adjusted to a cell concentration of 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS and 10 μM HEPES (HyClone Laboratories). Cells were analyzed by a flow cytometry (LSRFortessa, BD Biosciences). In each panel, at least 50,000 cells were analyzed.

Delivery of granzyme B from NK to K562 target cells was assessed using a GranToxiLux Kit (OncoImmunin). Briefly, K562 target cells were labeled with TFL-4 and a cell-permeable fluorogenic substrate. NK cells were then incubated with target cells. In this assay, as NK cells deliver granzyme B to target cells, granzyme B lyses the substrate, resulting in increased fluorescence in TFL-4+ target cells. Data acquisition and analysis were done by flow cytometry.

EZH2-targeted short hairpin RNAs and scrambled short hairpin RNA were kind gifts form Prof. Wang (Peking University First Hospital, Beijing, China) (14). Viral packaging and concentration was done by GeneChem. For transduction of human peripheral blood NK cells, cells were maintained in RPMI 1640 complete medium (mentioned above) for at least 3 d, with daily addition of cytokines (IL-2, 1000 U/ml; IL-21, 20 ng/ml). At a density of 2–5 × 105, NK cells were inoculated into 24-well plates, and a multiplicity of infection of 30–50 lentivirus, 8 μg/ml of protamine sulfate (Sigma-Aldrich), and 6 μM BX795 (InvivoGen) were added to the wells. The plate was centrifuged at 32°C and 1500 × g for 1.5 h. The infected cells were incubated overnight at 37°C and 5% CO2. The supernatant was then removed, and fresh complete medium was added. After 3 d of expansion, EGFP+ NK cells were sorted by flow cytometry (FACSAria II; BD Biosciences). For NK92MI cell transduction, cells were maintained in α-MEM complete medium (mentioned above) for at least 3 d, with daily addition of cytokines (IL-21, 20 ng/ml). At a density of 2–5 × 105, NK92MI cells were inoculated into 24-well plates, and a lentiviral multiplicity of infection of 30–50, 8 μg/ml polybrene (Sigma-Aldrich), and 6 μM BX795 (InvivoGen) were added to the wells. The plate was then centrifuged at 32°C and 1500 × g for 1.5 h. Infected cells were incubated overnight at 37°C and 5% CO2. The supernatant was removed, and fresh complete medium was added. After expansion for 3 d, EGFP+ NK92MI cells were evaluated by flow cytometry (FACSAria II; BD Biosciences). Transduction efficiency is shown in Supplemental Fig. 2.

Cellular extracts were prepared by incubating the cells in a RIPA buffer solution (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP40, 1 mM PMSF, and 1× protease inhibitor mixture) for 30 min at 4°C. Protein concentrations were determined using the BCA Assay Kit (Thermo Fisher Scientific). Cell extracts (10 μg) were denatured for 5 min in 5× SDS-PAGE loading buffer, separated on SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated at 4°C overnight in the presence of appropriate Abs, followed by incubation with secondary anti-mouse or anti-rabbit Abs (Cell Signaling Technology). Bands were visualized using the ECL Chemiluminescence Detection Kit (Vazyme Biotech), according to the manufacturer’s instructions. The Abs used in this experiment were as follows: anti-Histone H3 (4499; Cell Signaling Technology), anti–Tri-Methyl Histone H3 (9733; Cell Signaling Technology), anti-EZH2 (5246; Cell Signaling Technology), anti–β-actin (sc-8432; Santa Cruz Biotechnology), and anti-PKD2 (sc-28331; Santa Cruz Biotechnology).

This experiment was performed as previously described (13). The primers used in this study are shown in Supplemental Table I.

Total RNA was extracted using TRIzol Reagent (Invitrogen) and assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies) and Qubit Fluorometer (Invitrogen). Briefly, libraries were prepared using 1 μg of total RNA and the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs). Libraries were subjected to paired-end sequencing with pair end 150-bp reading length on an Illumina NovaSeq sequencer (Illumina). Clean data were mapped to hg19 reference genome using the STAR software (v2.7.9). Expression quantification of genome-wide genes was performed using htseq-count (v0.13.5). Differential expression analysis was performed using the edgeR R package (v3.34.0). With a cutoff value of |log2 (fold change)| > 1 and a false discovery rate (FDR) < 0.05, differential expression genes (DEGs) were identified in UNC1999-treated and control NK92MI cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were separately performed on identified DEGs using the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov/). GO and KEGG terms with p ≤ 0.05 and n > 5 were selected. For Gene Set Enrichment Analysis (GSEA), gene lists derived from RNA-sequencing data were compared with gene lists in the publicly available Molecular Signatures Database v7.4. GSEA statistical analysis was performed using GSEA v4.1.0 for Mac. Gene sets with |a normalized enrichment score |> 1, p < 0.05, and FDR < 0.25 were considered significantly enriched (15). Raw files are deposited at the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) database under accession number GSE184127.

This assay was performed as previously described (13). Supplemental Table I shows the primers used in this experiment.

Chromatin immunoprecipitation-sequencing (ChIP-seq) data were downloaded from the Roadmap Epigenomics Project (ftp://ftp.ncbi.nlm.nih.gov/pub/geo/DATA/roadmapepigenomics/). Wig files were downloaded and converted to bigWig for visualization in an integrative genomics viewer.

The small-molecule inhibitor UNC1999 is specific for the histone methylases EZH2 and EZH1. In addition, it reduces the level of H3K27me3 modification in the NK92MI cell line in a time- (Fig. 1A) and concentration-dependent manner [described in (13)]. In this study, NK92MI cells were treated with 10 µM UNC1999 for 48 h and then incubated with target K562 cells for 2 h. The results showed that the NK92MI cells in the UNC1999 treatment group had significantly higher degranulation [CD107a is a marker for NK cell degranulation, described in (16)] levels than the control group (Fig. 1C). To verify this result, we treated NK92MI cells with another small-molecule inhibitor, GSK343 (specific for histone methylase EZH2), and found that GSK343 also upregulated the degranulation level of NK92MI cells (Fig. 1C). Besides activating NK cells with the target cell K562, we also activated NK92MI cells with PMA/ionomycin (PMA/Iono), and the target cell activation results were consistent. Both small-molecule inhibitors (UNC1999 and GSK343) upregulated the degranulation levels of NK92MI cells (Fig. 1D). Finally, we knocked down the expression of EZH2 in NK92MI cells (Fig. 1B, Supplemental Fig. 2) and found that, consistent with the results of small-molecule inhibitors, the degranulation level of NK92MI cells in the knockdown group was significantly higher than the control group (Fig. 1E).

FIGURE 1.

Inhibition of EZH2 enzyme activity or expression promoted NK cell degranulation. (A) Western blot was used to detect changes in H3K27me3 modification status in NK92MI cells treated with DMSO (control) and UNC1999. Data are presented as mean ± SD for n = 3. Statistical analyses were performed using one-way ANOVA. ***p < 0.01. (B) Western blot was used to evaluate changes in EZH2 expression and H3K27me3 modification status of NK92MI cells with or without EZH2 knockdown. Data is presented as mean ± SD for n = 3. Statistical analyses were performed using one-way ANOVA. ***p < 0.01. (C) NK92MI cells and (F) human peripheral blood NK cells were treated with DMSO (control) and two small-molecule inhibitors at specified concentrations for 48 h. Treated effector cells were incubated for 2 h with K562 target cells. Flow cytometry was used to evaluate degranulation levels of effector cells. (D) NK92MI cells and (G) human peripheral blood NK cells were treated with DMSO (control) and two small-molecule inhibitors at specified concentrations for 48 h. Treated effector cells were activated with PMA/Iono for 2 h. Flow cytometry was used to evaluate degranulation levels in effector cells. Lentivirus infection knocked down EZH2 in (E) NK92MI cells and (H) human peripheral blood NK cells. Treated effector cells were activated for 2 h with PMA/Iono. Flow cytometry was used to evaluate degranulation levels of effector cells. Statistical analyses were performed using one-way ANOVA. Error bars represent standard deviations of results from nine samples in three independent experiments. ***p < 0.01.

FIGURE 1.

Inhibition of EZH2 enzyme activity or expression promoted NK cell degranulation. (A) Western blot was used to detect changes in H3K27me3 modification status in NK92MI cells treated with DMSO (control) and UNC1999. Data are presented as mean ± SD for n = 3. Statistical analyses were performed using one-way ANOVA. ***p < 0.01. (B) Western blot was used to evaluate changes in EZH2 expression and H3K27me3 modification status of NK92MI cells with or without EZH2 knockdown. Data is presented as mean ± SD for n = 3. Statistical analyses were performed using one-way ANOVA. ***p < 0.01. (C) NK92MI cells and (F) human peripheral blood NK cells were treated with DMSO (control) and two small-molecule inhibitors at specified concentrations for 48 h. Treated effector cells were incubated for 2 h with K562 target cells. Flow cytometry was used to evaluate degranulation levels of effector cells. (D) NK92MI cells and (G) human peripheral blood NK cells were treated with DMSO (control) and two small-molecule inhibitors at specified concentrations for 48 h. Treated effector cells were activated with PMA/Iono for 2 h. Flow cytometry was used to evaluate degranulation levels in effector cells. Lentivirus infection knocked down EZH2 in (E) NK92MI cells and (H) human peripheral blood NK cells. Treated effector cells were activated for 2 h with PMA/Iono. Flow cytometry was used to evaluate degranulation levels of effector cells. Statistical analyses were performed using one-way ANOVA. Error bars represent standard deviations of results from nine samples in three independent experiments. ***p < 0.01.

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We then repeated the above experiments in human peripheral blood NK cells. Consistent with our expectations, inhibiting the enzymatic activity of EZH2 or knocking down the expression of EZH2 in NK cells upregulated the degranulation level of NK cells (Fig. 1B, F–H; Supplemental Fig. 2).

Normally, the degranulation ability of NK cells is positively correlated with the cytotoxicity of NK. Therefore, we tested the killing ability of NK cells after being treated with small-molecule inhibitors. NK92MI cells were treated with the specified concentrations of small-molecule inhibitors (UNC1999 or GSK343) for 48 h before incubation with target cell K562 for 4 h.

Contrary to the expected results, we found that the killing ability of NK92MI cells treated with UNC1999 or GSK343 was significantly lower than that of the control group (Fig. 2A, 2B). To further validate this result, we knocked down the expression of EZH2 in NK92MI cells and noticed that the depletion of EZH2 resulted in the reduction of the NK cells killing ability (Fig. 2C). Finally, we repeated the above experiments in human peripheral blood NK cells, and the results are consistent (Fig. 2D–F).

FIGURE 2.

Inhibition of EZH2 enzyme activities or expression of downregulated NK cell cytotoxicity. (A) NK92MI cells and (D) human peripheral blood NK cells were treated with DMSO (control) and small-molecule inhibitor (UNC1999) at specified concentrations for 48 h. The treated effector cells were incubated with K562 target cells for 4 h. Flow cytometry was used to evaluate the killing capacity of effector cells. (B) NK92MI cells and (E) human peripheral blood NK cells were treated with DMSO (control) and a small-molecule inhibitor, GSK343, at specified concentrations for 48 h. The treated effector cells were incubated with K562 target cells for 4 h. Flow cytometry was used to evaluate the killing capacity of effector cells. Lentiviral infection knocked down the expression of EZH2 in (C) NK92MI cells and (F) human peripheral blood NK cells. Effector cells were incubated with K562 target cells for 4 h. Flow cytometry was used to assess the killing capacity of effector cells. GraphPad was used to calculate the area under the curve values for the killing curve, and one-way ANOVA analysis performed to compare the area under the curve values. The p values of less than 0.05 were considered significant. Experiments are repeated at least twice *p < 0.05, ***p < 0.01.

FIGURE 2.

Inhibition of EZH2 enzyme activities or expression of downregulated NK cell cytotoxicity. (A) NK92MI cells and (D) human peripheral blood NK cells were treated with DMSO (control) and small-molecule inhibitor (UNC1999) at specified concentrations for 48 h. The treated effector cells were incubated with K562 target cells for 4 h. Flow cytometry was used to evaluate the killing capacity of effector cells. (B) NK92MI cells and (E) human peripheral blood NK cells were treated with DMSO (control) and a small-molecule inhibitor, GSK343, at specified concentrations for 48 h. The treated effector cells were incubated with K562 target cells for 4 h. Flow cytometry was used to evaluate the killing capacity of effector cells. Lentiviral infection knocked down the expression of EZH2 in (C) NK92MI cells and (F) human peripheral blood NK cells. Effector cells were incubated with K562 target cells for 4 h. Flow cytometry was used to assess the killing capacity of effector cells. GraphPad was used to calculate the area under the curve values for the killing curve, and one-way ANOVA analysis performed to compare the area under the curve values. The p values of less than 0.05 were considered significant. Experiments are repeated at least twice *p < 0.05, ***p < 0.01.

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To further examine the effect of EZH2 on NK92MI cell killing ability, we observed the killing ability of the UNC1999 group, the GSK343 group, and the control group in a real-time way (real-time cytotoxicity assay). We observed that, in the early stages of killing (0–4 h), the target cells killed by the UNC1999 group and GSK343 group were significantly less than the control group, whereas there was no significant difference in the number of target cells killed between the groups at the late stage of killing (8–12 h) (Fig. 3A). At the same time, we used the traditional killing method (cytotoxicity assay) to detect the killing efficiency (NK92MI versus HeLa, NK92MI versus K562, and peripheral blood NK versus K562) after incubation for 12 h, and the results of the UNC1999 group and the GSK343 group were similar to the results of control group (Fig. 3B–D).

FIGURE 3.

Inhibition of EZH2 enzyme activity did not affect the long-term killing ability of NK cells. (A) The RTCA analyzer was used to evaluate the long-term killing ability of NK92MI cells. Time is presented on the horizontal axis, and cell indices (CI values) are presented on the vertical axis. The CI value is directly proportional to the number of living cells. The higher the number of live cells, the higher the CI value. We used GraphPad to calculate the area under the curve values for 0–4 h early stages of killing period for each group and performed one-way ANOVA on the area under the curve values. The p values of less than 0.05 were considered significant. Experiments were repeated twice. NK92MI cells were treated with DMSO (control) and small-molecule inhibitors UNC1999 and GSE343 at specified concentrations for 48 h. Treated effector cells were incubated with (B) HeLa or (C) K562 target cells for 12 h. Flow cytometry was used to detect the killing ability of effector cells. (D) Peripheral blood NK cells were treated with DMSO (control) and the small-molecule inhibitors UNC1999 and GSE343 at specified concentrations for 48 h. Treated effector cells were incubated with K562 target cells for 12 h. Flow cytometry was used to assess the killing ability of effector cells. GraphPad was used to calculate the area under the curve values for the killing curve, and one-way ANOVA was performed to compare the area under the curve values. The p values of less than 0.05 were considered significant. Experiments are repeated at least twice.

FIGURE 3.

Inhibition of EZH2 enzyme activity did not affect the long-term killing ability of NK cells. (A) The RTCA analyzer was used to evaluate the long-term killing ability of NK92MI cells. Time is presented on the horizontal axis, and cell indices (CI values) are presented on the vertical axis. The CI value is directly proportional to the number of living cells. The higher the number of live cells, the higher the CI value. We used GraphPad to calculate the area under the curve values for 0–4 h early stages of killing period for each group and performed one-way ANOVA on the area under the curve values. The p values of less than 0.05 were considered significant. Experiments were repeated twice. NK92MI cells were treated with DMSO (control) and small-molecule inhibitors UNC1999 and GSE343 at specified concentrations for 48 h. Treated effector cells were incubated with (B) HeLa or (C) K562 target cells for 12 h. Flow cytometry was used to detect the killing ability of effector cells. (D) Peripheral blood NK cells were treated with DMSO (control) and the small-molecule inhibitors UNC1999 and GSE343 at specified concentrations for 48 h. Treated effector cells were incubated with K562 target cells for 12 h. Flow cytometry was used to assess the killing ability of effector cells. GraphPad was used to calculate the area under the curve values for the killing curve, and one-way ANOVA was performed to compare the area under the curve values. The p values of less than 0.05 were considered significant. Experiments are repeated at least twice.

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Killing tumor cells by NK cells is a very complex process affected by many factors. NK cells exhibit increased degranulation levels after being treated with small-molecule inhibitors, although their killing ability is reduced. It is suggested that small-molecule inhibitors may affect the binding ability of NK cells to target cells, the formation of IS, or intracellular toxic particles polarization. We used flow cytometry to detect the binding ability of NK cells to target cells (Fig. 4A), and the ability of NK cells to form immune synaptic and toxic particle polarization was observed by laser confocal microscopy (Fig. 4B, 4C). However, UNC1999 did not affect the binding ability of NK cells, the formation of NK cell IS, and the polarization of NK cells (Fig. 4D). What is more, the intracellular expression of perforin and granzyme B were not changed in the control group compared with the UNC1999 group (Supplemental Fig. 1).

FIGURE 4.

Inhibition of EZH2 enzyme activity did not affect the binding of NK cells to target cells, formation of IS, and polarization of toxic particles. (A) Allophycocyanin–Cy7–stained NK92MI effector cells were incubated with CFSE-labeled K562 target cells for a specified time. Flow cytometry was used for detecting the binding capacity of NK92MI cells to K562 target cells treated with or without small-molecule inhibitors. (B) NK92MI cells were treated with DMSO (control) or the small-molecule inhibitor UNC1999 and incubated with CFSE-stained K562 target cells for 30 min, respectively (lower panel). Typical synapse between NK92MI and K562 is shown in the upper panel. Confocal microscopy was performed to observe the ability of effector cells to form an accumulation of F-actin. (C) NK92MI cells were treated with DMSO (control) or a small-molecule inhibitor UNC1999 and incubated with K562 target cells for 30 min. Anti-perforin (green) and anti–α-tubulin (red) staining were done to locate perforin and microtubule-organizing center. (D) Quantitation of conjugates, polarization, and synapse were performed, respectively. One hundred cell–cell contacts were analyzed in (B) and (C) for statistical analysis. Statistical analyses were performed using two-tailed unpaired t tests. Error bars represent SDs from the mean for three independent experiments.

FIGURE 4.

Inhibition of EZH2 enzyme activity did not affect the binding of NK cells to target cells, formation of IS, and polarization of toxic particles. (A) Allophycocyanin–Cy7–stained NK92MI effector cells were incubated with CFSE-labeled K562 target cells for a specified time. Flow cytometry was used for detecting the binding capacity of NK92MI cells to K562 target cells treated with or without small-molecule inhibitors. (B) NK92MI cells were treated with DMSO (control) or the small-molecule inhibitor UNC1999 and incubated with CFSE-stained K562 target cells for 30 min, respectively (lower panel). Typical synapse between NK92MI and K562 is shown in the upper panel. Confocal microscopy was performed to observe the ability of effector cells to form an accumulation of F-actin. (C) NK92MI cells were treated with DMSO (control) or a small-molecule inhibitor UNC1999 and incubated with K562 target cells for 30 min. Anti-perforin (green) and anti–α-tubulin (red) staining were done to locate perforin and microtubule-organizing center. (D) Quantitation of conjugates, polarization, and synapse were performed, respectively. One hundred cell–cell contacts were analyzed in (B) and (C) for statistical analysis. Statistical analyses were performed using two-tailed unpaired t tests. Error bars represent SDs from the mean for three independent experiments.

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Schwarz et al. (6) hypothesized that when the influx of calcium ions in NK cells increased abnormal toxic granules of NK cells would be released. For instance, when an NK cell contains six toxic particles, the NK cell releases the first two toxic particles to the first target cell, then releases the next two toxic particles to the second target cell, then releases the last two toxic particles to the third target cell. However, when the calcium influx of NK cells increases, NK cells may release all the six toxic particles to the first target cell at once, resulting in only the first target cell receiving toxic particles and eventually being killed by NK cells.

Based on the above assumptions, we examined the flow of calcium ions in NK cells. Initially, calcium flow significantly increased in NK92MI cells under PMA/Iono stimulation after being treated with different concentrations of UNC1999 (Fig. 5A). Calcium flow changes in NK cells are mainly determined by two different processes, including calcium ions stored in the ER of NK cells being released into the cytoplasm or calcium ions in microenvironment flowing into cytoplasm through the calcium channel on the cell membrane. To distinguish which process was affected by small-molecule inhibitors, we chelated the calcium ions in the microenvironment with 2 mM EGTA. It was found that the calcium ion flux in UNC1999 group was still significantly higher than the control group, indicating that UNC1999 increases the storage of calcium ions in the ER (Fig. 5B). To further confirm this result, we activated NK92MI cells with anti-NKp46 and anti-2B4 instead of PMA/Iono. Regardless of the presence or absence of calcium ions in the microenvironment, the calcium ion flux of the UNC1999 group was significantly higher than the control group (Fig. 5C, 5D). Last, we repeated the above experiments in human peripheral blood NK cells. Collectively, these results suggest that the inhibition of EZH2 enzymatic activity by UNC1999 can upregulate calcium ion storage in the ER of human peripheral blood NK cells and further increase calcium flux (Fig. 5E–H).

FIGURE 5.

EZH2 activity-dependent upregulation of calcium ions induced abnormal toxic particle release from NK cells. (A) NK92MI cells and (E) human peripheral blood NK cells were treated with DMSO (control) or small-molecule inhibitor UNC1999 at the specified concentrations for 48 h. Effector cells were collected in 2 mM Ca2+–PBS. Flow cytometry was used to evaluate calcium flux of effector cells under PMA/Iono stimulation. (B) NK92MI cells and (F) human peripheral blood NK cells were treated with DMSO (control) or the small-molecule inhibitor UNC1999 at the specified concentrations for 48 h, and the effector cells were collected in 2 mM EGTA–PBS. Flow cytometry was used to assess calcium flux of effector cells under PMA/Iono stimulation. (C) NK92MI cells and (G) human peripheral blood NK cells were treated with DMSO (control) or small-molecule inhibitor UNC1999 at the specified concentrations for 48 h, and the effector cells were collected in 2 mM Ca2+–PBS. Flow cytometry was used to assess calcium flux of effector cells stimulated by anti-NKp46/anti-2B4. (D) NK92MI cells and (H) human peripheral blood NK cells were treated with DMSO (control) or the small-molecule inhibitor UNC1999 at the specified concentrations for 48 h, and effector cells were collected in 2 mM EGTA–PBS. Flow cytometry was used to measure calcium flux in effector cells stimulated by anti-NKp46/anti-2B4. All experiments were repeated at least twice.

FIGURE 5.

EZH2 activity-dependent upregulation of calcium ions induced abnormal toxic particle release from NK cells. (A) NK92MI cells and (E) human peripheral blood NK cells were treated with DMSO (control) or small-molecule inhibitor UNC1999 at the specified concentrations for 48 h. Effector cells were collected in 2 mM Ca2+–PBS. Flow cytometry was used to evaluate calcium flux of effector cells under PMA/Iono stimulation. (B) NK92MI cells and (F) human peripheral blood NK cells were treated with DMSO (control) or the small-molecule inhibitor UNC1999 at the specified concentrations for 48 h, and the effector cells were collected in 2 mM EGTA–PBS. Flow cytometry was used to assess calcium flux of effector cells under PMA/Iono stimulation. (C) NK92MI cells and (G) human peripheral blood NK cells were treated with DMSO (control) or small-molecule inhibitor UNC1999 at the specified concentrations for 48 h, and the effector cells were collected in 2 mM Ca2+–PBS. Flow cytometry was used to assess calcium flux of effector cells stimulated by anti-NKp46/anti-2B4. (D) NK92MI cells and (H) human peripheral blood NK cells were treated with DMSO (control) or the small-molecule inhibitor UNC1999 at the specified concentrations for 48 h, and effector cells were collected in 2 mM EGTA–PBS. Flow cytometry was used to measure calcium flux in effector cells stimulated by anti-NKp46/anti-2B4. All experiments were repeated at least twice.

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To investigate the above hypothesis, we examined the ability of NK92MI cells and human peripheral blood NK cells to deliver toxic particles granzyme B to target cells treated with or without UNC1999. The results revealed that fewer K562 cells in the UNC1999 group and GSK343 group obtained granzyme B after incubation with effector cells (Fig. 6A). We repeated the above experiments in human peripheral blood NK cells, and the results are consistent (Fig. 6B).

FIGURE 6.

Abnormal toxic particle release resulted in fewer target cells obtaining toxic particles. (A) NK92MI cells and (B) human peripheral blood NK cells were treated with DMSO (control) or small-molecule inhibitors UNC1999 and GSK343 at the specified concentrations for 48 h, and effector cells were incubated with TLF4-stained K562 target cells for 1.5 h. Flow cytometry was used to measure the efficiency of granzyme B delivery by effector cells. Error bars represent standard deviations from the mean for n = 3. Statistical analyses were performed using one-way ANOVA. *p < 0.05, ***p < 0.01.

FIGURE 6.

Abnormal toxic particle release resulted in fewer target cells obtaining toxic particles. (A) NK92MI cells and (B) human peripheral blood NK cells were treated with DMSO (control) or small-molecule inhibitors UNC1999 and GSK343 at the specified concentrations for 48 h, and effector cells were incubated with TLF4-stained K562 target cells for 1.5 h. Flow cytometry was used to measure the efficiency of granzyme B delivery by effector cells. Error bars represent standard deviations from the mean for n = 3. Statistical analyses were performed using one-way ANOVA. *p < 0.05, ***p < 0.01.

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To further determine the molecular mechanism by which UNC1999 upregulates NK cell calcium flux, we performed second-generation high-throughput sequencing analysis of UNC1999-treated and control NK92MI cells. We found that 354 genes were upregulated and 109 genes were downregulated in NK92MI cells treated with UNC1999 (Fig. 7A; top 200 DEGs were list in Supplemental Table II). This result was consistent with the transcriptional repression function of the PRC2 complex. GO and KEGG pathway analysis were performed on DEGs. GO terms associated with nucleosome assembly, positive regulation of gene expression, and metal ion binding (Fig. 7E). KEGG pathway analysis showed that the PI3K–Akt signaling pathway, the Hippo signaling pathway, and Jak-STAT signaling pathway were significant (Fig. 7F). Subsequently, we performed GSEA analysis to determine whether a defined set of genes showed significant and consistent differences compared with our data. Consistent with expectations, the UNC1999-treated group positively correlated with the KODON_EZH2_TARGET gene set. In addition, EZH2 inhibition was significantly associated with the ION_CHANNEL_BINDING gene set. A survey of all genes in the ION_CHANNEL_BINDING gene set revealed that PKD2, a calcium permeable cation channel involved in calcium transport and calcium signaling, was the most highly induced ion channel gene (Fig. 7D).

FIGURE 7.

Identification of UNC1999-responsive gene signatures by high-throughput sequencing analysis. (A) Volcano plots show the DEGs screened by |log2 (fold change)| > 1 and FDR < 0.05 in transcriptome sequencing. Green dots represent upregulated genes, and red dots represent downregulated genes. GSEA analysis for EZH2_TARGETS (B) and ION_CHANNEL_BINDING (C) gene sets. (D) Heatmap of 62 genes in the ION_CHANNEL_BINDING gene sets. (E) GO analysis results of DEGs in control versus UNC1999 groups. (F) KEGG pathway enrichment results of DEGs in control versus UNC1999 groups. Dot sizes represent the counts of DEGs in a pathway, and dot colors from red to blue indicate p values from low to high.

FIGURE 7.

Identification of UNC1999-responsive gene signatures by high-throughput sequencing analysis. (A) Volcano plots show the DEGs screened by |log2 (fold change)| > 1 and FDR < 0.05 in transcriptome sequencing. Green dots represent upregulated genes, and red dots represent downregulated genes. GSEA analysis for EZH2_TARGETS (B) and ION_CHANNEL_BINDING (C) gene sets. (D) Heatmap of 62 genes in the ION_CHANNEL_BINDING gene sets. (E) GO analysis results of DEGs in control versus UNC1999 groups. (F) KEGG pathway enrichment results of DEGs in control versus UNC1999 groups. Dot sizes represent the counts of DEGs in a pathway, and dot colors from red to blue indicate p values from low to high.

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Another small-molecule inhibitor, GSK-J4, specific for the histone demethylase JMJD3/UTX, can increased global levels of the repressive H3K27me3 mark. A high-throughput sequencing analysis of GSK-J4–treated and control NK cells was performed as previously described (17). In that study, the expression level of PKD2 was significantly downregulated (data not show), which is consistent with our data.

To verify the results of high-throughput sequencing, we treated NK92MI cells with 10 μM UNC1999 for 48 h. Real-time quantitative PCR (qPCR) and Western blot confirmed that the expression of calcium channel PKD2 was significantly upregulated at both the mRNA and protein levels (Fig. 8A, 8B). Subsequently, we downloaded and analyzed a series of ChIP-seq data in the Gene Expression Omnibus database, including ChIP-seq data for different histone modifications in human peripheral blood NK cells as well as ChIP-seq data of different cells (CD56+, CD4+, CD8+, and CD34+) for H3K27me3 modification. We observed a significant H3K27me3 modification alongside H3K4me1 and H3K4me3 modifications, indicating that PKD2 is in a “poised” state in NK cells at rest (Fig. 8C). Further, the promoter region of PKD2 in CD34+ hematopoietic stem cells, CD4+ T cells, and CD8+ T cells was not modified by H3K27me3, suggesting that EZH2 regulation of PKD2 is specific to NK cells (Fig. 8C). This result explains the lack of a significant change in calcium flux in T cells after being treated with UNC1999 in PBMCs (data not shown). Finally, we designed five pairs of primers in the promoter region of PKD2 and then analyzed the modification status of H3K27me3 in the promoter region of PKD2 by ChIP-qPCR. It was found that in the vicinity of the two pairs of primers c and d, the modification of H3K27me3 was significantly downregulated after being treated with UNC1999. This is an indication that EZH2-regulated PKD2 expression is directly dependent on the enzymatic activity of EZH2 (Fig. 8D).

FIGURE 8.

Inhibition of EZH2 activity upregulated the expression of calcium channel PKD2. (A) Real-time qPCR was used to evaluate the expression levels of PKD2 in NK92MI cells after being treated with DMSO (control) or the small-molecule inhibitor UNC1999. GAPDH was used as the internal reference. Statistical analyses were performed using two-tailed unpaired t tests. Error bars represent mean ± SD for three independent experiments. ***p < 0.01. (B) Western blot was used to quantify the expression of PKD2 at the protein level in NK92MI cells after being treated with DMSO (control) or different concentrations of small-molecule inhibitor UNC1999. β-actin was used as the internal reference. Statistical analyses were performed using one-way ANOVA. Error bars represent mean ± SD for three independent experiments. ***p < 0.01. (C) H3K4me1, H3K4me3, H3K36me3, H3K27ac, and H3K27me3 modified states in the PKD2 promoter region of CD56+ resting NK cells (in blue box). Modified states of H3K27me3 in the PKD2 promoter region of CD56+ NK resting cells, CD34+ hematopoietic stem cells, CD4+ T cells, and CD8+ T cells (in red box). (D) ChIP-qPCR of H3K27me3 across the promoter of PKD2 in NK92MI cells treated with DMSO or UNC1999 for 48 h. GAPDH and 5 PKD2 primers are alphabetically labeled on the x-axis. Results are represented as fold changes over control with GAPDH as the negative control. Data are presented as mean ± SD for n = 3. Statistical analyses were performed using two-tailed unpaired t tests. *p < 0.05. Experiments were repeated twice.

FIGURE 8.

Inhibition of EZH2 activity upregulated the expression of calcium channel PKD2. (A) Real-time qPCR was used to evaluate the expression levels of PKD2 in NK92MI cells after being treated with DMSO (control) or the small-molecule inhibitor UNC1999. GAPDH was used as the internal reference. Statistical analyses were performed using two-tailed unpaired t tests. Error bars represent mean ± SD for three independent experiments. ***p < 0.01. (B) Western blot was used to quantify the expression of PKD2 at the protein level in NK92MI cells after being treated with DMSO (control) or different concentrations of small-molecule inhibitor UNC1999. β-actin was used as the internal reference. Statistical analyses were performed using one-way ANOVA. Error bars represent mean ± SD for three independent experiments. ***p < 0.01. (C) H3K4me1, H3K4me3, H3K36me3, H3K27ac, and H3K27me3 modified states in the PKD2 promoter region of CD56+ resting NK cells (in blue box). Modified states of H3K27me3 in the PKD2 promoter region of CD56+ NK resting cells, CD34+ hematopoietic stem cells, CD4+ T cells, and CD8+ T cells (in red box). (D) ChIP-qPCR of H3K27me3 across the promoter of PKD2 in NK92MI cells treated with DMSO or UNC1999 for 48 h. GAPDH and 5 PKD2 primers are alphabetically labeled on the x-axis. Results are represented as fold changes over control with GAPDH as the negative control. Data are presented as mean ± SD for n = 3. Statistical analyses were performed using two-tailed unpaired t tests. *p < 0.05. Experiments were repeated twice.

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Normally, the degranulation ability of NK cells is positively correlated with the cytotoxicity of NK cells. However, in our results, it was observed that the NK cells treated with the small-molecule inhibitors UNC1999 or GSK343 had a significantly enhanced degranulation capacity, although with a reduced lethality. Thomas et al. (18) reported that i.v. Ig can induce fatigue of NK cells, which further induces degranulation and IFN-γ expression but inhibits their cytotoxic activity, suggesting that our findings could be related to NK cell fatigue. However, in our results, we neither observed changes in IFN-γ expression nor changes of genes related to NK cells fatigue.

NK cells and target cells form a special area at the point of contact, known as the IS, through which toxic particles are delivered into the target cells. Upon recognition of a target cell, various receptors and signal molecules on the surface of the NK cell quickly aggregate at the center of the IS to form a supramolecular activation cluster (19, 20), which then initiates actin recombination. Under three-dimensional confocal microscopy, it was observed that actin quickly formed a high-density ring structure at the contact position for the transport of toxic particles (20, 21). At the molecular level, the formation of the IS requires the collaboration of several activating receptors such as integrin and NKG2D. Its downstream molecule Cdc42 is activated, and signals are transmitted to WASp, which is directly responsible for the polymerization of actin (2224). In addition to the reorganization of microtubules and actin cytoskeleton, another key step is the transport of cytotoxic particles. Cytotoxic particles initially polymerize along the microtubules to the microtubule-organizing center before moving to the IS with the aid of the molecular motor dynein (25, 26). Finally, the motor protein nonmuscle myosin IIA regulates the connection between F-actin and cytotoxic particles, allowing cytotoxic particles to pass through the F-actin network to the cell membrane (27, 28). These reports suggest that the abnormality of NK cell degranulation and killing effects may be related to the formation of the IS and the polarization of toxic particles. Therefore, using confocal microscopy, we observed NK cell IS formation and toxic particle polarization of NK cells treated with the small-molecule inhibitors (UNC1999 or GSK343). However, we did not observe abnormalities in IS formation and polarization of toxic particles, an indication that the reduced cytotoxicity in NK cells is not associated with the above processes.

Polarization and degranulation in NK cells are independent processes, and the interaction of activating receptor LFA1 with its ligand ICAM-1 is sufficient to induce cytotoxic particles polarization of NK (29). Once the cytotoxic particles have completed polarization and reached the cell membrane, docking and priming begins. GTPase Rab27a plays an important role in the docking of toxic particles (30). Rab27a deficiency in mice has been shown to induce Griscelli syndrome, an immunodeficiency disease which significantly reduces CTL cytotoxicity (31). After cytotoxic particles are docked, Munc13-4 regulates the priming CTL cells and NK cells. Degranulation is a complex and delicate process (32), and functional studies have also found that mutations in syntaxin11 and Munc18-2 and vesicle-associated proteins SNAREs, VAMP7, VAMP8, and VAMP4 can cause degranulation defects (5). This implies that these vesicle-associated proteins play important roles in the process of degranulation. However, our transcriptome sequencing results did not reveal any abnormal expression of the above genes, indicating that other factors could be responsible for the abnormal increase in degranulation.

In addition, Ca2+ influx is a necessary factor to ensure the normal progress of degranulation. Upon the activation of NK cells, the downstream signaling molecule PLC γ is activated and hydrolyzes PIP2 to generate the second messenger IP3, which in turn mediates Ca2+ transport from the ER to the cytoplasm. Meanwhile, the calcium ion sensor STIM detects the lack of calcium ions and activates the calcium ion channel ORAI1 on the cell membrane, therefore commencing a store-operated Ca2+ entry that triggers calcium ions to flow from the microenvironment into the cytoplasm (2, 33). Patients with mutations of STIM1 or ORAI1 have been reported to exhibit severe immunodeficiency (7, 34). Both types of patients exhibited a degranulation defect, whereas polarization of NK cells was normal. These results further illustrated the importance of the calcium signal in the degranulation process. Schwarz et al. (6) hypothesized that increased influx of calcium ions in NK cells leads to release of abnormal toxic granules of NK cells. For instance, when an NK cell contains six toxic particles, the NK cell releases the first two toxic particles to the first target cell, the next two toxic particles to the second target cell, then releases the last two toxic particles to the third target cell. However, when the calcium influx of NK cells increases, NK cells may release all six toxic particles to the first target cell at once, which causes death of the first target cell. Based on the above assumptions, we measured the Ca2+ flow in NK cells. We observed that regardless of the presence or absence of Ca2+ in the microenvironment, the Ca2+ flux in the inhibitor group was significantly higher than the control group, indicating that the induced calcium signal in NK cells upregulates the Ca2+ storage in the ER of NK cells. Consequently, NK cells release a large number of toxic particles to a small number of target cells within a short period of time and eventually show a temporary decrease in killing capacity.

We performed transcriptome sequencing on both NK cells in UNC1999 and control groups. PKD2, which encodes a multi-transmembrane channel protein and is usually involved in calcium transport and calcium signal transduction in renal epithelial cells, was identified. PKD2 is usually minimally expressed in NK cells. We observed significant H3K27me3 modification alongside H3K4me1 and H3K4me3 modifications in the PKD2 promoter region, indicating that PKD2 exists in a poised state in latent NK cells. This result suggests that the regulation of PKD2 expression by EZH2 is dependent on its methylase activity. Unlike NK cells, in CD34+ hematopoietic stem cells, CD4+ T cells, and CD8+ T cells, the promoter region of PKD2 does not have the specific modification of H3K27me3, suggesting that regulation of PKD2 expression by EZH2 is specific to NK cells [consistent with our previous research (3537)].

In this study, we coincidentally observed that induced calcium signal leads to unbalanced vesicle release that eventually inhibits their cytotoxic activity. This is an indication that the balanced calcium signal plays an important role in NK cell activity. Because histone modifications are characterized by accuracy, complexity, and reversibility and serve as potential drug targets, elucidation of its role in NK cell activity may provide novel ideas for cancer immune evasion or some hyperimmune diseases.

This work was supported by the National Natural Science Foundation of China Grants 81972652 (to X.W.), 81801638 (to Y.L.), and 31600705 (to J.Y.), the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (IDHT20190510 to X.W.), Ministry of Science and Technology of People’s Republic of China Grant 2014CB910100 (to X.W.), Tianjin Medical University General Hospital incubation foundation Grant ZYYFY2017006 (to Y.L.), and Natural Science Foundation of Tianjin City (Tianjin Natural Science Foundation) (Grant 17JCQNJC09000 (to J.Y.).

The raw files presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) under accession number GSE184127.

The online version of this article contains supplemental material.

Abbreviations used in this article

ChIP-seq

chromatin immunoprecipitation-sequencing

DEG

differential expression gene

ER

endoplasmic reticulum

FDR

false discovery rate

GO

Gene Ontology

GSEA

Gene Set Enrichment Analysis

IS

immune synapse

KEGG

Kyoto Encyclopedia of Genes and Genomes

PMA/Iono

PMA/ionomycin

qPCR

quantitative PCR

RTCA

Real-Time Cellular Analyzer

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The authors have no financial conflicts of interest.

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