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. 2018 Aug 2;9:1771. doi: 10.3389/fimmu.2018.01771

Figure 6.

Figure 6

PAG-KO bone marrow-derived mast cells (BMMCs) with CSK-KD exhibit enhanced degranulation, calcium response, and migration toward chemoattractants, but reduced adhesion to fibronectin and production of TNF-α. (A) β-glucuronidase release was determined in IgE-sensitized PAG-KO BMMCs with CSK-KD (CSK-KD/PAG-KO) or pLKO.1/PAG-KO or pLKO.1/WT control cells activated (30 min) or not with various concentrations of antigen. (B) The presence of CD107a on the cell surface was analyzed by flow cytometry in IgE-sensitized and antigen-activated (10 min) CSK-KD/PAG-KO BMMCs and appropriate controls. (C) Calcium response examined in CSK-KD/PAG-KO BMMCs or pLKO.1/PAG-KO or pLKO.1/WT control cells. IgE-sensitized cells were loaded with Fura-2, exposed to antigen (100 ng/ml; arrow), and Fura-2 fluorescence was analyzed for 300 s. Statistical significance of differences between CSK-KD/PAG-KO versus pLKO.1/PAG-KO is denoted by asterisks and between CSK-KD/PAG-KO versus pLKO.1/WT are denoted by hashtags. The data in (A–C) represent means ± SEM calculated from 4–7 independent experiments performed in duplicates or triplicates. (D) IgE internalization in PAG-KO BMMCs with CSK-KD or control pLKO.1 cells. The IgE-sensitized cells were activated with antigen (500 ng/ml) for various time intervals and fixed with 4% paraformaldehyde. IgE was quantified as described in Figure 1F. Means ± SEM calculated from three independent experiments are shown. (E,F) Adhesion of CSK-KD/PAG-KO BMMCs or control cells to fibronectin-coated surfaces. The cells were sensitized with IgE, loaded with calcein and activated with various concentrations of antigen (E) or SCF (F). Fluorescence was determined before and after washing out non-adherent cells, and the percentages of adherent cells were calculated. (G) Chemotaxis of BMMCs with PAG-KO, CSK-KD/PAG-KO, or wild-type (WT) cells was determined in Boyden chambers. The cells were sensitized with IgE and their migration toward antigen (250 ng/ml) and SCF (50 ng/ml) was determined. The results in E, F, and G represent means ± SEM from five independent experiments. (H) IgE-sensitized BMMCs with CSK-KD/PAG-KO or pLKO.1/PAG-KO or pLKO.1/WT control cells were activated or not for 6 h with antigen (100 ng/ml) and concentrations of TNF-α secreted into the supernatants were determined. Means ± SEM were calculated from four independent experiments. Statistical significance of intergroup differences was determined using one-way ANOVA with Tukey’s post-test (A,B,E–H) or two-way ANOVA with Bonferroni post-test (C,D). * and #P < 0.05; ** and ##P < 0.01; and *** and ###P < 0.001.