Fig. 3: Loss of granule acidity prevents effects of lysosomotropic challenge.
From: Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production
a–b Granule acidification in mefloquine-treated mast cells. Mast cells were incubated ± mefloquine (20 µM) for 30 min or bafilomycin-A1 (BAF; 20 nM) for 3 h. Additionally, NAC-pretreated mast cells were incubated with mefloquine (20 µM) for 30 min. Cells were subsequently incubated with LysoSensor Blue DND-167 (1 μM) for 1 h (a) or acridine orange (5 μg/mL) for 15 min (b) and granule acidification was assessed using flow cytometry. Data are expressed as means ± SEM. c Representative Western blot analysis of mMCP6. Mast cells were preincubated with NAC (8 mM) for 2 h or BAF (20 nM) for 3 h or left untreated, followed by incubation with mefloquine (20 μM). Cells treated with either PBS, NAC or BAF alone were included as controls. At the indicated time points, cytosolic extracts were prepared and subjected to Western blot analysis for presence of the granule protease mMCP6. d Quantitative analysis of mMCP6 expression. e Effect of BAF on mefloquine-induced ROS generation. Mast cells were preincubated with NAC (8 mM) for 2 h or BAF (20 nM) for 3 h or left untreated, followed by incubation with mefloquine (20 μM). Cells treated with either PBS, NAC or BAF were included as controls. After 30 min, cells were washed and incubated with CM-H2DCFDA for 30 min. Subsequently, cells were washed and cellular ROS levels were assessed by flow cytometry. The bars represent means ± SEM of the gMFI for CM-H2DCFDA. f Effect of BAF on mefloquine-induced cell death in mast cells. Mast cells were preincubated with BAF (20 nM) for 3 h or left untreated, followed by incubation of cells with mefloquine (20 μM). Cells treated with either PBS, NAC or BAF were included as controls. After 2 h, cells were washed and stained with Annexin V and DRAQ7 and cell death was assessed by flow cytometry. Data are expressed as means ± SEM (n = 3)
