Fig. 3: XKR8-mediated extensive phospholipid re-shuffling facilitates NETs formation.
a–c, PS exposure in WT, XKR8 KO, 2DA KI (XKR82DA) BMNs treated with PMA and NET formation treated with PMA (100 nM, 6 h), A23187 (1.25 μM, 4 h), nigericin (5 μM, 4 h) or LPS (10 μg ml−1, 2 h). Representative histogram (a) and quantification (b) of PS exposure in live cells (annexin V+PI−) (n = 3), and quantification of NET formation (c, n is shown on the graphs). d,e, Quantification of NETs in neutrophil-like HL-60 cells: WT, XKR8 KO and XKR8 KO cells reconstituted with full-length mouse XKR8 (KO + XKR8FL) (d), and XKR8 mutants (2DA and S/T-3A) (e) following PMA (100 nM, 6 h) treatment (n = 8). f, Immunoblot analysis of cleavage of XKR8 during PMA-induced NET formation. Mouse XKR8 (XKR8FL) and XKR82DA mutant were GFP-tagged and expressed in XKR8 KO dHL-60 cells. β-Actin was a loading control. g, Quantification of NETs in neutrophil-like HL-60 WT, XKR8 KO and XKR8 KO cells reconstituted with XKR8 mutants D30A and W45A. NETs were induced with PMA (100 nM, 6 h) (n = 8). h,i, PS and PE exposure during NET formation in WT and XKR8 KO BMNs treated with PMA (100 nM, 3 h), detected using annexin V-APC and duramycin-Cy3 staining, respectively. Representative flow cytometry plot (h) and quantification (i) of PS+/PE+ in live cells (PI−) are shown (n = 3). j, Quantification of non-extractable NBD-labeled phospholipids (PLs) translocated by activated XKR8 during PMA-induced NET formation (n = 3). Bovine serum albumin (BSA; 5 mg ml−1) was used to back-exchange the NBD-PLs from the outer layer of the PM. PC, phosphatidylcholine; SM, sphingomyelin. NETs were determined by the area of chromatin DNA (c–e,g). Each dot represents an individual field of view (c–e,g) or a biological replicate (b,i,j). Data are represented as mean ± s.e.m. from one representative experiment of three (a–c,f) and four (d,e,g,h–j) independent experiments. Statistical analysis was performed using a two-way ANOVA (b–e,g,i,j). P values are shown on the graphs.
