Inhibition of IgE-induced mast cell histamine release, cysLT generation and TNF synthesis by various inhibitors.

<p>Inhibition of IgE-induced mast cell histamine release, cysLT generation and TNF synthesis by various inhibitors.</p

IgE alone stimulates mast cells to cysLT synthesis.

<p>Mast cells were incubated with different concentrations of IgE (white bars) for 1 h. Anti-IgE-induced cysLT secretion (black bar) is presented as a positive control. Results are shown as the mean ± SEM of four independent experiments and each experiment was carried out in duplicate (n = 4). ***p<0.001.</p

IgE alone stimulates phosphorylation of ERK, p38 and PI3K in mast cells.

<p>Mast cells were treated with IgE alone at 5 µg/mL for the indicated times. Total cell lysates (50 µg) were subjected to Western blotting analysis using anti-phospho antibodies specific to ERK1/2 (pERK1/2), p38 (pp38) or PI3K (pPI3K). The same blots were reprobed with respective antibodies that recognize these signaling molecules, irrespective of the phosphorylation states. Results are representative of 3 independent experiments.</p

IgE alone up-regulates FcεRI expression on rat mast cells.

<p>Mast cells were incubated with IgE at 1 µg/mL (white bars) and 5 µg/mL (black bars) or without IgE (control FcεRI expression on native mast cells referred to as 100%) for 1, 6 and 24 h. Surface FcεRI expression was analyzed by flow cytometry using mouse anti-rat FcεRI primary antibodies (10 µg/mL) and FITC-labeled goat anti-mouse secondary antibodies (5 µg/mL) (A). Representative flow cytometry histogram showing FcεRI expression on native mast cells as well as on mast cells treated with 5 µg/mL for 1 h (B). Results are presented as the mean ± SEM of four independent experiments (n = 4). *p<0.05.</p

IgE alone induces <i>de novo</i> TNF synthesis in mast cells.

<p>Mast cells were incubated with different concentrations of IgE (white bars) for 6 h. Anti-IgE-induced TNF secretion (black bar) is shown as a positive control (A). For TNF mRNA assessment mast cells were incubated with IgE (white bars) or anti-IgE (black bars) both at 5 µg/mL for the indicated times (B). TNF mRNA levels were determined by qRT-PCR and presented as fold-increase over the value of cytokine mRNA expression in non-stimulated cells after normalization with the transcript level of the housekeeping gene rat Actb. Results are presented as the mean ± SEM of at least three separate experiments performed in duplicate (A) or triplicate (B) (n≥3). *p<0.05, **p<0.01, ***p<0.001.</p

IgE alone affects mast cell migration.

<p>Mast cells were pretreated with IgE at 1 µg/mL or 5 µg/mL (IgE-coated mast cells) or medium (native mast cells) for 1 h before being placed into the upper wells, whereas medium (spontaneous migration), CCL5 or TNF had been added to lower wells of Boyden microchamber. Results are presented as the mean ± SEM of at least four independent experiments and each experiment was carried out in duplicate (n≥4). ***p<0.001.</p

Cell signaling inhibitors influence IgE-affected both spontaneous and induced mast cell migration.

<p>Mast cells were preincubated with U73122 (0.1 µM), Src I-1 (0.01 µM), LY294002 (50 µM) or medium alone for 1 h (none). Then, mast cells were treated with IgE at 5 µg/mL, washed and spontaneous (A), CCL5-induced (B) and TNF-induced (C) migration was examined in a Boyden microchamber. Bars for the positive controls demonstrate the same data set as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079286#pone-0079286-g004" target="_blank">figure 4</a>. Results are presented as the mean ± SEM of four independent experiments and each experiment was carried out in duplicate (n = 4). *p<0.05, **p<0.01, ***p<0.001.</p

IgE alone induces mast cell degranulation.

<p>Mast cells were incubated with different concentrations of IgE (white bars) for 1 h. Anti-IgE-induced histamine release (black bar) is shown as a positive control (A). Mast cells were stimulated with IgE at 5 µg/mL for indicated times of incubation (B). Results are presented as the mean ± SEM of at least four independent experiments and each experiment was carried out in duplicate (n≥4). *p<0.05, **p<0.01.</p

The response of mitochondrial pH to the inhibition of an efflux and the release component of (Ca²⁺)_c elevations.

<p>(A) The average effect of extracellular Ca²⁺ removal and its subsequent restitution on (Ca²⁺)_c and pH_mito changes in n = 18, n = 12, n = 21 cells for C, _2, _3 lines respectively, pretreated for 20 min with thapsigargin and 2-APB. The insets show SERCA-independent (Ca²⁺)_c clearance (upper) and pH_mito recovery (lower). (B) The average effect of cadmium (VDCCs inhibitor) in n = 29, n = 19, n = 22 cells for C, _2, _3 lines respectively, on KCl-evoked (Ca²⁺)_c influx and concomitant pH_mito changes. (C) The average effect of 5 mM La³⁺ in n = 16 cells for C, _2, _3 lines showing delay in (Ca²⁺)_c clearance and pH_mito alkalization under low extracellular Na⁺. (D) The average effect of extracellular alkaline pH (9.0) followed by pH return to 7.4 in n = 20, n = 12, n = 20 cells for C, _2, _3 lines respectively, on (Ca²⁺)_c elevations and pH_mito changes.</p

Single cell characterization of cellular pH in steady-state conditions.

<p>Average resting pH_mito and pH_cyto measured by simultaneous imaging of mitoSypHer and SNARF fluorescence, respectively. * P<0.05, pH_mito vs. pH_cyto within each line; ^# P<0.05, PMCA-deficient lines vs. control cells.</p