Fig. 4: Endogenous S100A12 induced neutrophil extracellular traps (NETs) formation by calcium influx overload.

a, b Intracellular calcium concentration detected by confocal Rhod2 staining, and statistical results after transfection of si-S100A12 or pcDNA3.1-S100A12 (n = 4 biological replicates). Scale bar, 10 μm. c Colorimetric detection of intracellular Ca²⁺ concentration in dHL-60 cells transfected with pcDNA3.1-S100A12 or si-S100A12 (n = 4 biological replicates). d, e Neutrophils transfected with or without si-S100A12 were preloaded with calcium probe Fluo-4 AM dye and incubated for 30 min in HBSS calcium-free media. After washing, cells were resuspended in Assay Buffer and stimulated with ionomycin (5 µM). Time-course of fluorescent signal was recorded with excitation and emission wavelengths of 488 and 526 nm, respectively. The fluorescence was normalized to the baseline fluorescence F0 (fluorescence measurement at the first time point). The Area under curve (AUC) was estimated using sums-of-squares method (n = 6 biological replicates). f–j Representative blots of peptidylarginine deiminase 4 (PAD4), p-CaMKII, myeloperoxidase (MPO), citrullination of histone 3 (citH3) and quantification in dHL-60 cells treated with pcDNA3.1-S100A12 and different concentration of calcium channel blocker (Verapamil) (n = 4 biological replicates). a: P < 0.0001 vs pcDNA3.1-NC + 0 µm Verapamil. k, l Mice were intraperitoneally injected with corn oil or Verapamil (5 mg/kg/d) every other day from Day 1 before the MI operation to post-MI Day 3. Survival curves and rates of WT and TG mice treated with Verapamil for 14 days after MI were analyzed using Log-rank (Mantel-cox) test. m, n Echocardiographic parameters were analyzed in WT and TG mice treated with verapamil at post-MI Day 28 (n = 5 biological replicates for WT MI group, n = 6 biological replicates for WT + Verapamil group, n = 3 biological replicates for TG MI group and n = 5 biological replicates for TG + Verapamil group). Data are expressed as mean ± SEM. Statistical analyses were carried out using two-tailed unpaired t-test for (m), (n), one-way ANOVA with Tukey’s multiple comparisons test for (e), (g), (h), (i), (j) and Brown-Forsythe and Welch’s ANOVA test for (b), (c). Source data are provided as a Source Data file.