Extended Data Fig. 7: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death.
From: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
a, SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b, Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in Fig. 3j. c, Confocal immunofluorescence micrograph to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan). The dashed line indicates the cross-section view represented in Fig. 3k. d, e, SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Two-sided Mann–Whitney test. e, Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t-test. f, g, SMCs were incubated with histone H4. f, Extracellular ATP. n = 5 biological replicates. Two-sided Mann–Whitney test. g, Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t-test. h, Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann–Whitney test. i, j, SMCs were incubated with recombinant histone H4 after pre-incubation with oleylamine or cholesterol sulfate (chl sulfate). i, Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide–membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, −), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. **P = 0.007; ***P = 0.0001 vs ctrl. j, Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, *P = 0.001; **P = 0.004. Data are mean ± s.d.
