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. 2023 May 10;14:1154108. doi: 10.3389/fimmu.2023.1154108

Figure 4.

Figure 4

MITF silencing reduces degranulation and Ca2+ influx. (A) LAD2 cells were transduced with control NT (non-target) shRNA or MITF shRNA 2 or MITF shRNA 3, and the efficiency of MITF silencing was assessed by western blot. α-Tubulin was used as a loading control. The unpaired t-test was used for statistical analysis (**p < 0.01*, ****p < 0.0001). Experiments are the mean ± SEM (n=4). (B) β-hexosaminidase was performed with different concentrations of SP and P+I (PMA+ionomycin, 10 ng/mL+0.5 μM) as a positive control in NT and MITF-silenced cells, n=4. (C) Degranulation was determined by flow cytometry using CD63 staining in unstimulated and SP (2uM) in the alive population (propidium iodide negative staining) in NT and MITF-silenced cells. (D) NT and MITF-silenced LAD2 cells were preincubated with Fluo4 and activated with SP (2µM) or ionomycin. A two-way ANOVA test was used for statistical analysis (**p<0.01, ***p<0.001, ****p<0.0001). Experiments are the mean ± SEM (n=3).