Fig 2: Stable expression of MrgX2 in HMC-1 cells enhances LL-37-internalization. The control plasmid-transfected HMC-1 cells (Control group) and MrgX2-expressing HMC-1 cells (MrgX2-transfected group) were incubated with phycoerythrin-conjugated anti-MrgX2 mouse mAbs, and (A) analyzed by flow cytometry. (B) Control plasmid-transfected HMC-1 cells (Control, left panel) and MrgX2-expressing HMC-1 cells (MrgX2, right panel) were cytocentrifuged following incubation with LL-37 (5 µM) at 37°C for 40 min, and LL-37 was stained with anti-LL-37 rabbit pAbs followed by anti-rabbit IgG-Alexa Fluor 488 (green staining). Nuclei were stained with DAPI (blue staining). Images are representative of 6 independent experiments. Magnification, ×400. (C) Control plasmid-transfected HMC-1 cells (Control group) and MrgX2-expressing HMC-1 cells (MrgX2 group) were incubated with or without LL-37 (5 µM) at 37°C for 40 min. The internalization of LL-37 was determined by measuring the fluorescence intensities using ImageJ software, and represented as a ratio of control plasmid-transfected HMC-1 cells (Control) incubated with LL-37 (5 µM). Data are presented as the mean ± standard deviation of 6 independent experiments. m/pAbs, mono/polyclonal antibodies; MrgX2, Mas-related gene X2.

Fig 3: Stable expression of MrgX2 in 293 cells enhances LL-37-internalization. (A) The control plasmid-transfected 293 cells (Control group) and MrgX2-expressing 293 cells (MrgX2-transfected group) were incubated with phycoerythrin-conjugated anti-MrgX2 mouse mAbs, and analyzed by flow cytometry. (B) Control plasmid-transfected 293 cells (Control, left panel) and MrgX2-expressing 293 cells (MrgX2, right panel) were cytocentrifuged following incubation with LL-37 (5 µM) at 37°C for 40 min, and LL-37 was stained with anti-LL-37 rabbit pAbs followed by anti-rabbit Immunoglobulin G-Alexa Fluor 488 (green staining). Nuclei were stained with DAPI (blue staining). Images are representative of 6 independent experiments. Magnification, ×400. (C) Control plasmid-transfected 293 cells (Control group) and MrgX2-expressing 293 cells (MrgX2 group) were incubated with or without LL-37 (5 µM) at 37°C for 40 min. The internalization of LL-37 was determined by measuring the fluorescence intensities using ImageJ software, and represented as a ratio of control plasmid-transfected 293 cells (Control) incubated with LL-37 (5 µM). Data are presented as the mean ± standard error of the mean of 6 independent experiments. ***P<0.001, as indicated. m/pAbs, mono/polyclonal antibodies; MrgX2, Mas-related gene X2.

Fig 4: Effects of neuraminidase treatment and endocytosis inhibitors on the degranulation and LL-37 internalization of LAD2 cells. LAD2 cells were treated with neuraminidase (0.2 U/ml, +Neu) or without (Control) at 37°C for 40 min, and further incubated with or without LL-37 (2 µM) for 40 min. (A) Thereafter, the release of ß-hexosaminidase was measured. Alternatively, the cells were cytocentrifuged, and LL-37 was stained with anti-LL-37 rabbit pAbs followed by anti-rabbit IgG-Alexa Fluor 488. (B) The internalization of LL-37 was determined by measuring the fluorescence intensities using ImageJ software, and represented as a ratio of the control cells (Control) incubated with LL-37 (2 µM) but without neuraminidase treatment. (C) In addition, LAD2 cells were treated with or without genistein (10 µM), and dynasore (10 µM) or chlorpromazine (2.5 µM), and further incubated with or without LL-37 (5 µM) at 37°C for 40 min, and the ß-hexosaminidase release was measured. Alternatively, the cells were cytocentrifuged, and LL-37 was stained with anti-LL-37 rabbit pAbs followed by anti-rabbit IgG-Alexa Fluor 488. (D) The internalization of LL-37 was determined by measuring the fluorescence intensities using ImageJ software, and represented as a ratio of the cells incubated with LL-37 (5 µM) but without endocytosis inhibitors. Data are presented as the mean ± standard error of the mean of 4 to 8 independent experiments. *P<0.05 and ***P<0.001, as indicated. IgG, immunoglobulin G; pAbs, polyclonal antibodies; N.D, not detected; MrgX2, Mas-related gene X2.

Fig 5: MrgX2 knockdown reduces degranulation and LL-37 internalization. Mrgx2 was knocked-down by MrgX2-targeted siRNA transfection, and confirmed by western blotting. The expression of GAPDH was evaluated as the internal control. Images are representative of 3 independent experiments. (A) MrgX2 expression was quantified by densitometry, and the expression in MrgX2-knockdown LAD2 cells (MrgX2 KD group) was expressed as a ratio of that in the control siRNA-transfected cells (Control group). (B) The control siRNA-transfected LAD2 cells (Control group) and MrgX2-knockdown cells (MrgX2 KD group) were incubated with or without LL-37 (1–2 µM) at 37°C for 40 min, and the release of ß-hexosaminidase was measured. (C) The control siRNA-transfected LAD2 cells (Control group, left panel) and MrgX2-knockdown cells (MrgX2 KD group, right panel) were cytocentrifuged following incubation with LL-37 (5 µM) at 37°C for 40 min, and LL-37 was stained with anti-LL-37 rabbit polyclonal antibodies followed by anti-rabbit Immunoglobulin-G-Alexa Fluor 488 (green staining). Nuclei were stained with DAPI (blue staining). Images are representative of 3 independent experiments. Magnification, ×400. (D) The control siRNA-transfected LAD2 cells (Control group) and MrgX2-knockdown cells (MrgX2 KD group) were incubated with or without LL-37 (1–2 µM) at 37°C for 40 min. The internalization of LL-37 was determined by measuring the fluorescence intensities using ImageJ software, and represented as a ratio of the control siRNA-transfected LAD2 cells (Control group) incubated with LL-37 (2 µM). Data are presented as the mean ± standard error of the mean of 3 independent experiments. MrgX2, Mas-related gene X2; siRNA, small interfering RNA; KD, knockdown.