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Keywords:

  • small molecular inhibitors;
  • cell activation;
  • TCR signaling;
  • transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Small molecular inhibitors are excellent tools for manipulating cell reactions. They are widely used in scientific research to study molecular mechanisms of cells under physiological and pathophysiological conditions as well as in clinical applications to treat patients. However, their selectivity is often not well known. Moreover, it can vary according to cell types and the analysis methods used. Therefore, it is usually not possible to make comparisons between the data presented in the literature. Here we analyzed the selectivity of five chosen inhibitors of calcineurin/NFAT activation under the same conditions. Using a combination of fluorescent cell barcoding and phospho-specific flow cytometry we studied the inhibition of activation of NF-κBp65 and MAPK pathways in stimulated primary human Th cells. This semi-high throughput approach enabled us to demonstrate that (i) CsA and NCI3 are around 5 to 10- and 20-fold less potent, respectively, at inhibiting phosphorylation of NF-κBp65 and p38 than activation of NFAT, (ii) AM404 is at least 15-fold selective for NFAT but already toxic at concentrations above 40 μM, (iii) INCA6 is not selective at all, and (iv) BTP1 is at least 100-fold selective for inhibition of NFAT activation relative to NF-κBp65, p38 and ERK1/2 phosphorylation. Altogether, our results not only show the applicability of a semi-high throughput inhibitor test system but also that BTP1 is the most selective inhibitor of calcineurin/NFAT activation among the studied inhibitors under the used conditions.

Ca2+/calcineurin/NFAT signaling is one of the main pathways triggered after antigen-specific stimulation of Th cells or simulation of this process using either antiCD3/CD28 antibodies or the chemicals PMA/ionomycin. Calcium/calmodulin-dependent activation of the Ser/Thr phosphatase calcineurin is a prerequisite for the concerted dephosphorylation and subsequent nuclear translocation of the transcription factors of the NFATc family. Binding of NFAT to its target promoter sequences induces the synthesis of such important cytokines as IL-2 and IFNγ (1, 2) as well as of surface molecules such as CD25, CD154 (CD40L), and CD134 (OX40) (3). Manipulation of NFAT activation by targeted inhibition is therefore not only of general interest for treating autoimmune reactions but also for identifying molecular pathomechanisms in inflamed tissue.

In clinical contexts, the cyclic undecapeptide Cyclosporine A (CsA) is widely used for pharmacological inhibition of immune reactions during transplantations or treatment of atopic dermatitis (4, 5). In complex with immunophilins, CsA effectively blocks the enzymatic activity of calcineurin (6), which is especially important in inhibiting memory Th cells (7–10). However, long-term treatment with CsA in high dosages is associated with severe side effects such as nephrotoxicity, carcinogenicity, and induction of hypertension/hyperkalemia (11, 12).

CsA is of limited value in scientific research because it has been shown that CsA inhibits not only NFAT activation but also the activation of NF-κB and AP-1 (13–17). Because of these non-selective properties of CsA, we and others searched for new and more specific calcineurin/NFAT inhibitors as potential alternatives to CsA. Here we selected four of these novel inhibitors: AM404, BTP1, NCI3, and INCA6. Each of them caused impaired dephosphorylation and nuclear translocation of NFAT (18–20). While the molecular mechanism underlying AM404 and BTP1 (19, 20) mediated NFAT inhibition remains to be analyzed, NCI3 and INCA6 (18, 21) were shown to target the association of calcineurin/NFAT by masking their interaction domain without blocking the phosphatase activity of calcineurin. The different modes of action of these inhibitors make it highly probable that they could interfere in different ways with the activation of NF-κB and AP1.

To study and compare the pathway selectivity of inhibitors we used an approach combining fluorescent cell barcoding and phospho-specific flow cytometry (22, 23) in primary human Th cells. This allowed the simultaneous measurement of multiple samples with reduced measurement time and antibody consumption (23). Our data provide an overview about the selectivity of certain inhibitors of the calcineurin/NFAT signaling pathway and will help to select the appropriate inhibitor as well as the correct concentration for different scientific purposes. Therefore, phospho-flow coupled with fluorescent cell barcoding is a rapid screening method for determining inhibitor selectivity in primary cell systems.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Inhibitors

The small molecular inhibitors Bay 11-7082, SB 203580, and U0126 were purchased from biomol (Hamburg, Germany). AM404 was purchased from Sigma (Taufkirchen, Germany), CsA from AWD (Dresden, Germany), and INCA6 from Tocris Bioscience (Eching, Germany). BTP1 was synthesized according to the literature (24, 25). The obtained analytical data are: mp 195–197°C, 1H NMR (300 MHz, DMSO-d6): δ 7.61–7.66 (m, 4H), 7.83 (s, 1H), 7.99–8.03 (m, 4H), 10.66 (s, 1H). MS (ESI+) 434 (100%, M+1). These data correspond to the reported data (24, 25). NCI3 was synthesized according to standard procedures. Cyclization of ethyl benzoylacetate with 3-amino-5-methyl-4-phenyl-pyrazole furnished the 7-hydroxy-pyrazolopyrimidine core. Subsequent reaction with phosphorus oxychloride affords the corresponding chloro-derivative, which was reacted with 5-amino-pentanol to give the target compound 3,5-diphenyl-7-(5-hydroxypentylamino)-2-methyl-pyrazolo[1,5-a]pyrimidine NCI3. The obtained analytical data are: mp 213–214°C, 1H NMR (300 MHz, DMSO-d6): δ 8.21 (m, 2H), 7.88 (m, 2H), 7.32–7.49 (m, 5H), 7.26 (t, J =7.1, 1H), 6.71 (s, 1H), 3.53 (m, 2H), 3.42 (m, 2H), 2.60 (s, 3H), 1.70 (m, 2H), 1.48 (m, 4H) (Magdalena Karanik, PhD thesis, Humboldt-University, Berlin 2005).

Barcoding Dyes and Antibodies

The fluorescent cell barcoding dyes Pacific Blue succinimidyl ester (Invitrogen, Karlsruhe, Germany) and Pacific Orange succinimidyl ester (MoBiTec, Göttingen, Germany) were used to label cells. Phospho-flow cytometry was performed using the following antibodies (all from BD Biosciences Heidelberg, Germany): anti-phospho NF-κBp65 (Ser529) Alexa Fluor 488 (catalog number 55842, clone K10-895.12.50), anti-phospho p38 (Thr180/pTyr182) PE (catalog number 612565, clone 36/p38 (pT180/pY182)), and pERK1/2 (Thr202/Tyr204) Alexa Fluor 647 (catalog number 612593, clone 20A).

Cell Isolation and Stimulation

Blood from healthy volunteers (DRK, Berlin, Germany) was collected in accordance with the recommendations of the local ethics committee on human studies (Charité, Berlin, Germany). Th cells were isolated via positive CD4 magnetic cell sorting (MACS technology; Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) with a purity of at least 95%. After MACS separation the cells were washed once with media (RPMI containing 10% fetal calf serum, 10 mM glutamic acid, 10 μg ml−1 2-mercaptoethanol, 100 U ml−1 penicillin, and 100 ml streptomycin per 500 ml) and rested in media over night at 4°C. Prior to inhibitor treatment and stimulation the cells were adapted to 37°C for 1 h in the incubator.

Isolated Th cells (1 × 106) were pretreated with inhibitors or 0.3% DMSO (as control) for 30 min prior to stimulation with PMA (10 ng ml−1) and ionomycin (1 μg ml−1) (all Sigma–Aldrich, Taufkirchen, Germany). Inhibitor treatment and stimulation were performed in media as mentioned above.

Luciferase Reporter Gene Assay

We measured all inhibitors in a NFAT-dependent reporter gene assay under the same conditions as previously described (18). In brief, Jurkat T cells electroporated (Amaxa protocol; Amaxa, Cologne, Germany) in the presence of NFAT- or pGL3- luciferase reporter gene plasmids (1–1.5 μg) (Promega, Mannheim, Germany) were cultured in RPMI for 16 h at 37°C in 5% CO2. Aliquots of electroporated cells were preincubated with the respective inhibitor or 0.3% DMSO for 30 min, followed by stimulation with 10 ng ml−1 PMA + 1 μg ml−1 ionomycin or 100 ng ml−1 TNF-α for 5 h. The level of the extracted luciferase was determined after cell lysis by bioluminescence measurement (luciferase assay system of Promega). The constitutively active pGL3-luciferase plasmid was used to test toxic effects of the inhibitors.

Fluorescent Cell Barcoding and Cell Staining

Stimulated Th cells were washed once with ice cold PBS, fixed in PBS containing 2% paraformaldehyde (20 min, 25°C) and subsequently permeabilized with 95% methanol (30 min on ice) containing the respective concentration of the barcoding dyes (Supporting Information Table). The labeled cells were washed twice with PBS containing 1% BSA. The different samples (up to 9) were combined into one FACS tube (final volume 100 μl PBS supplemented with 1% BSA) and incubated with antibodies against phospho-p65 (1:10), p38 (1:10), and ERK1/2 (1:10) at 25°C for 45 min.

Phospho-Specific Flow Cytometry

Flow cytometric analysis was performed using the FACS Canto II flow cytometer (BD Biosciences, Heidelberg, Germany) with the following laser configuration: 405, 488, and 633 nm. Fixed and permeabilized Th cells were identified in the forward scatter (FSC) and side scatter (SSC). Pacific Blue was detected in the 450/50 nm channel (364 V) and Pacific Orange was detected in the 510/50 nm channel (379 V) both excited by the 405 nm laser. Alexa 488 was detected in the 530/30 nm channel (429 V) and PE was detected in the 585/42 nm channel (405 V) both excited by the 488 nm Laser. Alexa 647 was detected in 660/20 nm channel (542 V) excited by the 633 nm laser. Unstained and single stained cells were used as staining controls and unstimulated cells were used as control for the phosphorylation analysis. Software compensation was performed resulting in the following compensation matrix: PE—Alexa 488 15%; Pacific Blue—Pacific Orange 16.8%; Pacific Orange—Pacific Blue 3.25%.

The amount of 2 × 104 CD4 cells was recorded and subsequently exported as list-mode data file (FCS 3.0) and analyzed with the FlowJo software (version 7.2.5; Tree Star) applying biexponential transformation.

Fixed and permeabilized lymphocytes were gated using FSC/SSC pseudocolor dot plots. The average frequency of gated lymphocytes was 85% ± 0.3%. Single samples were identified in dot plots of pacific orange/pacific blue pseudocolor and gated according to the fluorescence intensity of the barcoding dyes. Half offset histogram overlays of every single population were used to display the amount of phosphorylation (Figs. 2A and 2B) and Geo Mean values were used for further calculations (Fig. 2B). Detailed gate boundaries and statistics for every single experiment are available upon request.

Definition and Scoring of the Inhibitory Capacity

We defined three threshold levels indicating the amount of inhibition: (1) no or low inhibition (≤10%), (2) intermediate inhibition (10–50%), and (3) strong inhibition (≥50%). Using a scoring algorithm we performed a case-by-case analysis of all obtained data and marked the results in the stacked columns (Fig. 3) and in Table 2 in green, yellow, and red.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Determination of IC50-Values of Five Different Inhibitors Using NFAT Reporter Gene Assays

In Table 1 we summarized the literature data about NFAT IC50 values of 5 selected calcineurin/NFAT inhibitors. To have a uniform baseline for determining selectivity we measured all five inhibitors in a NFAT-dependent reporter gene assay under the same conditions (18). Our obtained IC50 values were also placed into Table 1. We have got similar IC50 values for the inhibitors BTP1 and NCI3 compared to the given references. CsA and AM404 were around fourfold more active in our hands using the NFAT-reporter gene assay. However, INCA6 was sixfold less active. This could be due to the different assays used. Roehrl et al. took a binding assay and measured the replacement of the VIVIT peptide from calcineurin by the inhibitor (21). Our own IC50 value of BTP1 (4 nM) is a bit lower compared to that of the given reference (6 nM). However, Trevillyan et al. used another reporter (whole IL-2 promoter instead of NFAT alone). The dose response curve of BTP1 obtained in NFAT-reporter gene assays (n = 2) is placed as Supporting Information Figure 1.

Table 1. IC50 values of selected inhibitors of calcineurin/NFAT activation
InhibitorIC50ReferenceIC50 (own data)a
  • a

    a Jurkat T cells (NFAT-dependent reporter gene expression).

  • b

    b IL-2 production in primary human Th cells.

  • c

    c Jukat T cells (IL-2 promotor-dependent reporter gene expression).

  • d

    d Displacement of VIVIT peptide.

CsA4 nM(1)b1 nM
AM40410 μM(20)a2.4 μM
BTP16 nM(19)c4 nM
INCA60.8 μM(21)d5 μM
NCI32 μM(18)a1.4 μM

The Activation of the NF-κB and AP-1 Signaling Pathway can be Analyzed by Flow Cytometry

First we proved whether it is possible to analyze the stimulation-dependent phosphorylation of p65, p38, and ERK1/2 in primary human CD4+ Th cells. Strong stimulation induced phosphorylation was clearly detectable at p65 (Ser529), p38 (Thr108, Tyr182), and ERK1/2 (Thr202/Tyr204), as shown by histogram overlays (Fig. 1). Application of specific inhibitors of NF-κB (Bay 11-7082) and ERK1/2 activation (U0126) rescued the activation-induced shift of fluorescence intensities of the Th cells. The phosphorylation level of p65 was reduced by 95% (±4%) by 2 μM Bay 11-7082 and was completely blocked by 4 μM Bay 11-7082 (100% ± 7%). ERK1/2 phosphorylation was reduced by 82% with 10 μM U0126 and by 87% with 20 μM U0126. In contrast, only a slight reduction of p38 phosphorylation by the specific p38 inhibitor SB 203580 was observed. However, SB 203580 specifically inhibits the p38 MAP kinase activity and only partially interferes with the phosphorylation of p38 (26–28).

thumbnail image

Figure 1. Proof of principle of cytometric determination of p65, p38, and ERK1/2 phosphorylation. Isolated primary human CD4+ T cells were pre-incubated with specific inhibitors of p65 (Bay 11-7082), p38 (SB 203580), and ERK1/2 (U0126) activation or DMSO (as control) for 30 min before stimulation with PMA/ionomycin for 15 min. Subsequently, the cells were fixed, permeabilized, and stained with antibodies recognizing p65 pSer529 (Alexa 488), p38 pThr180/pTyr182 (PE), and pERK1/2 pThr202/pTyr204 (Alexa 647). Histogram overlays show one representative experiment out of three.

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CsA does not Exclusively Inhibit the Calcineurin/NFAT Pathway

We applied the fluorescent cell bar-coding technique (23) to shorten the cytometric measurement process and to reduce the amount of antibodies for measurement of the pathway selectivity of the five inhibitors. Two different bar-coding dyes (Pacific Blue and Pacific Orange) and different concentrations/combinations of them allowed the simultaneous measurement of up to 9 different samples at one time.

As an example we plotted the bar-coding and the flow cytometric measurement of one representative CsA experiment (Fig. 2). We combined five different CsA concentrations and two controls (stimulated and unstimulated cells). The bar-coding concentrations were optimized to enable the clear separation of the samples in the cytometer (Fig. 2A). The bar-coded samples were pooled, stained with phospho-specific antibodies against p65, p38, and ERK1/2 and measured by flow cytometry. Then, forward deconvolution was performed as described by Krutzik (23). Basically, the cells were analyzed for separation of the bar-coded populations into FSC/pacific blue or FSC/pacific orange. Subsequently, the gated populations were dissected and analyzed for p65, p38, and ERK1/2 phosphorylation (Fig. 2B, upper panel). Finally, geometric mean values were used to calculate the inhibition of phosphorylation relative to the stimulated control (Fig. 2B, lower panel).

thumbnail image

Figure 2. CsA does not only inhibit calcineurin/NFAT activation. Human CD4+ T cells were treated as in Figure 1. (A) In addition, after permeabilization the cells were labeled with different concentrations of the barcoding dyes Pacific Blue and Pacific Orange. Subsequently, all samples were combined and stained together. (B) After cytometric measurement the individual samples were gated according to their barcoding intensities and analyzed separately for phosphorylation of p65, p38, and ERK1/2. Geo Mean values were used to calculate the inhibition of phosphorylation relative to the stimulated control without inhibitor (lower panel). One representative experiment out of four is shown.

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CsA inhibits the phosphorylation of NF-κBp65 and p38 but is around 5- to 10-fold less potent inhibiting these processes than NFAT activation. However, it is difficult to make the determination since CsA does not inhibit the activation of NF-κBp65 and p38 above 50%. The CsA effect on ERK1/2 phosphorylation was negligible (Figs. 2B and 3, upper row). In general, we observed differences in inhibitory effects between individual donors as indicated by the standard deviation in Figure 3.

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Figure 3. BTP1 is at least 100-fold selective for inhibition of NFAT activation relative to p38 and p65 phosphorylation under the used conditions. Isolated primary human Th cells were pre-incubated with different concentrations of CsA, AM404, BTP1, NCI3, and INCA6. Fluorescent cell barcoding, antibody staining, and data deconvolution were performed as in Figure 2. Mean values and SD of relative inhibition of phosphorylation (n = 3 or more) are shown. Colored bars represent three threshold levels of inhibition: green = no or low (≤10%), yellow = medium (10–50%) red = high (≥50%). Arrows in the first column indicate the respective NFAT IC50 values for each of the inhibitors used.

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NCI3 has a Similar Pathway Selectivity as CsA

Using the same experimental setup as described above for CsA (Fig. 2), we analyzed the selectivity of four other calcineurin/NFAT inhibitors. To classify the inhibitors we defined three threshold levels indicating the amount of inhibition: (1) no or low inhibition (≤10%), (2) intermediate inhibition (10–50%), and (3) strong inhibition (≥50%).

We measured the inhibitory potential of NCI3 in the concentration range of 0.25 and 50 μM because at concentrations above 50 μM we observed toxic effects in the NFAT reporter gene assay. NCI3 is about 20-fold less potent in inhibiting phosphorylation of NF-κBp65 and p38 than activation of NFAT. However, NCI3 does not inhibit the ERK1/2 phosphorylation even at the highest concentration used (50 μM).

The Inhibitors INCA6 and AM404 have Different Selectivity but are Both Toxic

The inhibitors INCA6 and AM404 showed already toxic effects in Jurkat cells above concentrations of 20 and 40 μM, respectively. INCA6 is not selective at all. For AM404 we concluded that it is at least 15-fold less potent to inhibit the other three phosphorylation events compared to NFAT activation.

The Inhibitor BTP1 is Highly Selective for Inhibition of the Calcineurin/NFAT Pathway in Activated Th Cells

Trevillyan et al. discovered BTP1 by screening a chemical library using a high throughput reporter gene assay with the luciferase gene under the transcriptional control of a full-length IL-2 promoter (19). They showed that BTP1 inhibits NFAT dephosphorylation by a mechanism other than direct inhibition of calcineurin phosphatase activity. We showed that BTP1 does not inhibit all three tested phosphorylation events and showed no toxicity in the reporter assays. BTP1 is at least 100-fold selective for inhibition of NFAT activation relative to NF-κBp65, p38, and ERK1/2 phosphorylation.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Inhibitors are in general valuable tools for studying molecular processes in cells. Although many small molecular inhibitors have been the subject of publications, in many cases their activity in primary cells and their specificity among the Ser/Thr phosphatases, as well as their selectivity among the important pathways, have not been studied (29). Moreover, the experimental conditions used to determine their activity and selectivity are usually not comparable with regard to cell types, stimulatory agents, concentration ranges employed, and other factors. Table 1 reflects these aspects for small molecular inhibitors of calcineurin/NFAT activation, one key step in Th cell activation.

In this study we analyzed the pathway selectivity of calcineurin/NFAT inhibitors by monitoring other main signaling pathways during Th cell activation, namely NF-κB and MAPKs. The latter activate the transcription factors c-jun and c-fos. To this end, activation-induced phosphorylation of NF-κBp65 (Ser529), p38 (Thr108/Tyr182), and ERK1/2 (Thr202/Tyr204) was measured in the presence of different concentrations of the selected inhibitors, namely, CsA (1), AM404 (20), BTP1 (19), INCA6 (21), and NCI3 (18). So far, very few data are available concerning the selectivity of these inhibitors and almost all of them were tested exclusively in cell lines such as Jurkat cells rather than in primary cells. Here we used primary human Th cells magnetically sorted from blood of healthy donors.

Our own calcineurin/NFAT IC50 values of each inhibitor (Table 1) were used as references and inhibitor concentrations below, and in particular above, these values were tested. For the simultaneous measurement of phosphorylation levels of NF-κBp65, p38, and ERK1/2 at different inhibitor concentrations we used the fluorescent cell barcoding method. This semi-high throughput flow cytometry-based approach (23) permitted a fast and reproducible screening, despite high standard deviations due to donor variations. In Table 2 we summarized and color coded the results applying three threshold levels (1 = green, no or low inhibition; 2 = yellow, intermediate inhibition; 3 = red, strong inhibition). We discovered that BTP1 is the most selective inhibitor among the five tested. For CsA we confirmed previous results showing that this inhibitor not only blocks NFAT but in addition NF-κBp65 and p38 activation (13–16). Furthermore, we showed that CsA already interferes with immediate early events, namely the phosphorylation of p65 and the MAP kinase p38 within 20 min after stimulation of Th cells.

Table 2. Inhibition of p65, p38, and ERK1/2 phosphorylation by different inhibitors of calcineurin/NFAT activation at NFAT IC50 concentrations and at 10-fold NFAT IC50 concentrations
inline image

Here we studied just five inhibitors among the published 42 low molecular weight inhibitors and 13 protein and peptide inhibitors of calcineurin/NFAT activation (29). Thereby we realized that inhibitors' selectivity must be carefully examined under the same experimental conditions, in particular using primary cells, and that this can be achieved by studying early phosphorylation. Such an analysis is less time-consuming and permits an overview of possible activation of transcription factors. Nevertheless, inhibitors with the same mode of action and similar selectivity could point to a specific cross talk between signaling pathways. The strong sensitivity of NFAT and NF-κB activation toward CsA already helped us to identify that the Ser/Thr phosphatase calcineurin transiently dephosphorylates the adapter protein Bcl-10, which is a crucial event for NF-κB activation (17).

In summary, we demonstrate the usefulness of the simultaneous monitoring of early phosphorylation events in primary human Th cells. We contribute further to knowledge concerning the selectivity of few known NFAT inhibitors in primary human T cells and their applicable concentrations for obtaining selective calcineurin/NFAT inhibition.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

The authors thank Stefan Kröger for creating the scoring algorithm, all members of the Baumgrass's lab for discussion and helpful advices, and Toralf Kaiser and Jenny Kirsch for supporting the flow cytometry measurements.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
MIFlowCyt.doc523KSupporting Information: MIFlowCyt
CYTO_22204_sm_SuppFig1.eps341KSupplemental Fig. 1: Dose response curve of BTP1. Jurkat T cells transfected with a NFAT luciferase reporter plasmid were incubated with BTP1 or DMSO as control before the cells were stimulated with PMA/ionomycin for 5 h. The luciferase reporter gene assay was performed as described previously [Sieber et al. Eur J Immunol 2007;37:2617-2626]. Data were depicted as percent inhibition compared to the stimulated control sample without inhibitor. A four parameter curve fit was used to calculate the IC50 value. Data out of two independent experiments are shown as MW + SD.
CYTO_22204_sm_SupplTab.pdf127KSuppl. Tab. 1: Collection of all applied amounts of barcoding dyes and inhibitor concentrations

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