Fig 2: Peptide concentration controls digital NFAT1 nuclear translocation kinetics in a responder fraction of stimulated naive OT-I cells.(A) Representative flow cytometry histograms displaying a timecourse of NFAT1 staining in OT-I nuclei after coculture with bulk splenocytes pulsed with 10 pM of OVA peptide. (B) Representative flow cytometry histograms of NFAT1-positive OT-I nuclei after 45 min of coculture with bulk splenocytes pulsed with indicated dose of OVA peptide. (C) Plots depicting compiled timecourse results from two experiments measuring NFAT1 localization in OT-I nuclei after coculture with bulk splenocytes pulsed with the indicated dose of OVA peptide. (D) Representative histograms of three experiments performed show that EGTA addition causes NFAT1 to exit OT-I nuclei. OT-I cells were cocultured with bulk splenocytes pulsed with 100 pM of OVA peptide. After stimulation for 30 min to allow for nuclear translocation of NFAT1, 5 mM EGTA was added to culture, and samples were harvested after 0,30, 60, or 90 min. (E) Line plots depicting median fluorescence intensity (m.f.i.) of NFAT1 in isolated OT-I nuclei stimulated similarly to (D). After 30 min of stimulation with OVA peptide, either 5 mM EDTA, 5 mM EGTA, or 100 nM FK506 was added to culture media where indicated. Samples were collected 0, 15, 30, 60, and 90 min after adding inhibitors. In (A)-(E), OT-I nuclei were identified as CellTrace Violethi ß-tubulinlo

Fig 3: Titrated doses of ionomycin does not accurately induce a digital NFAT1 response in OT-I cells.(A) NFAT1 localization in OT-I nuclei after different doses of ionomycin over the course of 240 min. Representative histograms displaying NFAT1-positive nuclei with indicated dose of ionomycin present in the coculture media. Nuclei were identified as CellTrace Violethi ß-tubulinlo (B) For comparison, results of NFAT1-positive OT-I nuclei during coculture with bulk splenocytes pulsed with indicated dose of OVA peptide.

Fig 4: MCU-KO enhances NFAT nuclear translocation.A–C, HEK293 cells and their MCU-KO counterparts were transfected with an NFAT4-GFP construct, stimulated with 10 µM CCh, and nuclear translocation of NFAT4-GFP was monitored over time using a fluorescence microscope (A; see Experimental procedures, the scale bar represents 10 µm). The ratio of nuclear/total fluorescence of NFAT4-GFP (B) and difference between maximal and basal ratios (? NFAT4; C) are represented for both groups of cells and were statistically analyzed using a Mann–Whitney test where **p < 0.01. (D–F), ImageStream analysis of native NFAT1 nuclear translocation using flow cytometry in wildtype and MCU-KO Jurkat cells stimulated with 2 µM thapsigargin (Tg) for 30 min. Native NFAT1 was labeled with a fluorescently tagged specific antibody, and NFAT1 nuclear translocation was determined by colocalization of NFAT1 with DAPI nuclear staining (D). Histograms showing the distribution (E) and intensity (F) of nuclear NFAT1 fluorescence from n = 3 independent experiments with 10,000 cells/run in wildtype Jurkat and MCU-KO cells. The results of these experiments were analyzed using an unpaired t test where ****p < 0.0001. MCU, mitochondrial Ca2+ uniporter; NFAT, nuclear factor for activated T cells.

Fig 5: Isolation of stimulated OT-I nuclei and analysis via conventional flow cytometry.(A) Representative forward scatter (FSC) and side scatter (SSC) plot of OT-I nuclei isolated and fixed in sucrose buffer compared with OT-I whole cells. (B) Flow cytometry histograms comparing nuclei versus whole cells stained with indicated Abs or propidium iodide (P.I.). (C) Confocal images displaying NFAT1 localization in isolated OT-I nuclei compared with whole cells after 30 min of stimulation with or without 1 µg/ml ionomycin.