Fig. 3

LDs entered into lysosomes. a WT or Tas2r138 siRNA-transfected neutrophils were infected with PAO1 (MOI: 5:1) or AHL-12 (50 μM) stimulated for 2 h. The LDs (stained by Nile red) and LAMP2 (green) were detected by immunofluorescence imaging (arrows showing the typical proteins colocation). b WT or Tas2r138 siRNA-transfected neutrophils were infected with PAO1 (MOI: 5:1) or AHL-12 (50 µM) stimulated for 2 h. The expression of CTSB, CTSD, and β-actin was tested by western blotting. c Neutrophils were incubated with 20 µM leupeptin for 24 h to inhibit lysosome function. Then the cells were infected with PAO1 (MOI: 5:1) for 2 h. The LDs (stained by Nile red) and LAMP2 (green) expression were detected by immunofluorescence imaging (arrows show the colocation between LAMP2 and LDs decrease). d Neutrophils were incubated with 20 µM leupeptin for 6 h, then the cells were infected with PAO1 (MOI: 5:1) for 2 h. Cells were fixed by 4% paraformaldehyde and permeabilized with 0.2% Triton-X 100 for 15 min. The LDs were stained by Nile red for 2 h. After washed by PBS, the fluorescence intensity was measured by Bio-Tek fluorescence plate reader (530/25, 590/35). The results were expressed as the mean ± SD and the significant difference level between two groups was defined by one-way ANOVA with Tukey post hoc tests, *p < 0.05