Neutrophil-Derived IL-6 Potentially Drives Ferroptosis Resistance in B Cells in Lupus Kidney

Figure 3

Neutrophil-derived IL-6 induces ferroptosis resistance in lupus kidney B cells. (a) The correlation matrix from scRNA-seq data showed IL-6 level correlated to the expression level of IL6R, STAT3, and SLC7A11 (xCT). (b) Flow cytometry analysis showed blocking IL-6 enhanced ferrous ions (FerroOrange) in lupus kidney B cells, which could be rescued by ferrostatin 1 treatment. Neutrophil and B cells were cocultured in transwell plates, in which B cells were placed in the bottom. (c) Dot plot showed statistical analysis of frequency of FerroOrange+ B cells from coculturing B cells and neutrophils, with or without IL-6 blocking. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; NS: no significance. (d) Flow cytometry analysis showed blocking IL-6 enhanced lipid peroxidation (LiperFluo) in lupus kidney B cells, which could be rescued by ferrostatin 1 treatment neutrophil and B cells were cocultured in transwell plates, in which B cells were placed in the bottom. (e) Dot plot showed statistical analysis of frequency of LiperFluo+ B cells from coculturing B cells and neutrophils, with or without IL-6 blocking. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; NS: no significance. (f) Dot plot showed statistical analysis of intracellular GSSG/GSH ratio from cocultured B cells and neutrophils, with or without IL-6 blocking. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; NS: no significance. (g) Dot plot showed statistical analysis of intracellular GSH from cocultured B cells and neutrophils, with or without IL-6 blocking. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; NS: no significance. (h) Immunofluorescent imaging analysis showed sulfasalazine (xCT inhibitor) treatment enhanced ferrous ions (FerroOrange) in lupus kidney B cells. Neutrophil and B cells were cocultured in transwell plates, in which B cells were placed in the bottom. Dead cells were removed by counterstaining fixable viability dye. (i) Dot plot showed statistical analysis of the relative fluorescent intensity of FerroOrange from coculturing B cells and neutrophils, with or without sulfasalazine treatment. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; NS: no significance. (j) Immunofluorescent imaging analysis showed sulfasalazine (xCT inhibitor) treatment enhanced lipid peroxidation (LiperFluo) in lupus kidney B cells. Neutrophil and B cells were cocultured in transwell plates, in which B cells were placed in the bottom. Dead cells were removed by counterstaining fixable viability dye. (k) Dot plot showed statistical analysis of the relative fluorescent intensity of LiperFluo from coculturing B cells and neutrophils, with or without sulfasalazine treatment. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; ; NS: no significance. (l) Dot plot showed statistical analysis of intracellular GSSG/GSH ratio from cocultured B cells and neutrophils, with or without sulfasalazine treatment. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; ; NS: no significance. (m) Dot plot showed statistical analysis of intracellular GSH ratio from cocultured B cells and neutrophils, with or without sulfasalazine treatment. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. ; ; NS: no significance. (n) Flow cytometry analysis showed that lupus kidney B cell proliferation was inhibited after sulfasalazine treatment. Dead cells were removed by counterstaining fixable viability dye. (o) Dot plot showed statistical analysis of the proliferated B cell from coculturing B cells and neutrophils, with or without sulfasalazine treatment. 8 wells were in each group. Data represents similar results from at least 3 independent experiments. ; NS: no significance.

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