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. 2020 Mar 25;21(5):e48904. doi: 10.15252/embr.201948904

Figure 6. Potassium channel activity is required for Ca2+ responses and IFNγ production, but not hyperproliferation in EGR4KO T cells.

Figure 6

CD4+ T cells were isolated by negative selection from WT, EGR1−/−, or EGR4−/− mice.
  1. WT or EGR4−/− cells were loaded with fura‐2 AM and plated on anti‐CD3/CD28 antibodies for 2 h. Senicapoc (4 μM) or TRAM‐34 (5 μM) was added where marked by the arrow.
  2. CD4+ T cells were isolated by negative selection from WT or EGR4−/− mice and incubated with anti‐CD3/CD28 antibodies for 6 h in the presence or absence of either senicapoc, TRAM‐34, or Shk‐Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean ± SEM; three biological replicates were examined; and each biological replicate includes two technical replicates. ***P < 0.001; ****P < 0.0001.
  3. To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4−/− CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti‐TCRβ antibodies in the presence of vehicle, senicapoc (5 μM), or TRAM‐34 (5 μM) for 4 days and then collected for FACS analysis.
  4. Generation analysis was completed for all data using FlowJo software. Data are mean ± SEM; three biological replicates were examined; and each biological replicate includes two technical replicates.