Fig 2: Patient T cell responses to T cell receptor stimulation.(A) Representative flow plots showing expression of IFN-? in human PBMCs from a healthy donor and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr and cultured for further 4 days in the presence of IL-2 before re-stimulation with PMA plus ionomycin for 5 hr for cytokine analysis. Cells were gated for CD4+ T cells. (B) Quantitative mRNA expression analysis (± s.d.m.) of indicated cytokines from human PBMCs (cultured as mentioned above) with or without stimulation with PMA plus ionomycin for 5 hr. (C) Levels of IL-2 and TNF in human PBMCs from culture supernatants of cells stimulated as described above (B) were determined by ELISA. (D) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after transient stimulation with anti-CD3 antibody cross-linking, or ionomycin (0.5 µM) in the presence of external solution containing 2 mM Ca2+ (left) as indicated. Bar graphs show baseline subtracted ratio values for anti-CD3 antibody cross-linking or ionomycin (average± SEM) from six independent experiments (right). (E) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after store-depletion with thapsigargin (1 µM) stimulation in Ca2+-free Ringer’s solution. SOCE was measured by addition of 2 mM Ca2+-containing Ringer’s solution as indicated (left). Bar graph shows baseline subtracted ratio values at the peak of SOCE (average± SEM) from four independent experiments (right). (F) Phosphorylated JNK levels in CD4+ T cells from healthy control and patient PBMCs (cultured as indicated in A), stimulated with anti-CD3 antibody for indicated times. Bar graphs show average± SEM from three independent experiments. ** p < 0.005, *** p < 0.0001.

Fig 3: LiCl inhibits the activation of GSK-3ß/ß-catenin signal pathway in rat hippocampus. (A–D) Western blots results (A) and quantitative measurement of GSK-3ß (B), ß-catenin (C) and JNK (D) in rat hippocampus. Data are represented as mean ± SEM; n = 3; one-way ANOVA, F (2, 6) = 31.33; **P < 0.01 compared with control group; # P < 0.05 compared with SEV group.

Fig 4: SOCE and JNK phosphorylation in T cells expressing individual allelic mutations in CRACR2A.(A) Representative traces showing averaged SOCE from control (48 cells) or CRACR2A-KO Jurkat T cells (KO) transduced with an empty vector (55 cells) or those encoding FLAG-tagged WT CRACR2A (45 cells), CRACR2AE278D (E278D, 40 cells), or CRACR2AR144G/E300* (DM, 50 cells) mutants (left). Cells were stimulated with anti-CD3 antibodies, followed by ionomycin treatment in the presence of external solution containing 2 mM Ca2+. Bar graphs show averaged baseline subtracted peak SOCE (± s.e.m.) from anti-CD3 antibody and ionomycin treatments, from three independent experiments (right). (B) Representative traces showing averaged SOCE induced by thapsigargin (TG) treatment from control (52 cells) or CRACR2A-KO Jurkat T cells (KO) transduced with empty vector (46 cells) or those encoding FLAG-tagged WT CRACR2A (49 cells), CRACR2AE278D (E278D, 51 cells), or CRACR2AR144G/E300* (DM, 50 cells) mutants (top). Cells were stimulated with thapsigargin in Ca2+-free solution to deplete the intracellular stores and exposed to external solution containing 2.0 mM Ca2+. Bar graphs below show averaged baseline subtracted SOCE levels (± s.e.m.) at the peak (center) or later time point (sustained – 900 s, bottom) from three independent experiments. (C) Phosphorylated JNK levels in control or CRACR2A KO Jurkat T cells stably expressing WT and indicated mutants of CRACR2A, stimulated with anti-CD3 antibody for indicated times. Graphs show average± SDM from three independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.0001.

Fig 5: Application of different pump profiles and quantitative assays. (a) Overview of cell processes that have been investigated in kinetic environments. (b) Quantifying volume and (c) signal transduction of Hog1 nuclear localization over time in live single S. cerevisiae yeast cells exposed to an instant step increase to 0.4 M NaCl (solid line, 79 cells) or to a linear gradient of 0.4 M NaCl in 10 minutes (dashed line, 90 cells). (d) JNK phosphorylation over time measured with flow cytometry in human THP1 cells after exposure to an instant step increase to 0.1 M NaCl (solid line, 636,628 cells) or to a linear gradient of 0.1 M in 60 minutes (dashed line, 1,599,923 cells). (e) Single cell distributions of single-molecule RNA FISH measurements of STL1 mRNA in S. cerevisiae yeast cells exposed to an instant step increase to 0.4 M NaCl (solid line, 3269 cells) or a linear gradient of 0.4 M in 10 minutes (dashed line, 2164 cells). Thick lines are the mean and shaded area are the standard deviation from two or three biological replica experiments of single cells.