Fig 1: SPARC deficiency associates to a defective clearance by macrophages of dying PMN(A-I) (A) In vitro efferocytosis assay using WT peritoneal macrophages cultured 30 min with 1:1 ratio of WT and Sparc-/- peritoneal PMN or BM PMN (treated or not with anti-Fas Ab, to induce apoptosis, as control) (statistic: *** p < 0.001; ****p < 0.0001, Student's t test, mean ± SEM). The analysis highlights a specific defect in the clearance of Sparc-/- PerC PMN. (B) Percentage of F4/80hi and F4/80int peritoneal macrophages (Mf) engulfing CFSE-labeled apoptotic Jurkat cells. (C) Time-lapse confocal microscopy and relative quantification of WT and Sparc-/- macrophages (unstained, bright field) co-cultured with either WT or Sparc-/- macrophages (Mf, labeled with PKH26). Contrary to WT PMN that died and were almost completely engulfed by macrophages from both strains, Sparc-/- PMN accumulated in the co-culture without removal; scale bars, 10 µm. (D) Relative quantification; statistical analysis: one-way ANOVA, multiple comparisons test (Tukey). Mean ± SEM is shown; **p < 0.01; ***p < 0.01; ****p < 0.0001. (E) Expression of the “eat-me” (phosphatidylserine, PS, and calreticulin) and of (F) “don't-eat-me” (CD47 and CD31) signals in peritoneal WT and Sparc-/- PMN at day 130 after pristane treatment; statistical analysis: one-way ANOVA, multiple comparison test (Tukey). *p < 0.05; **p < 0.01; ****p < 0.0001; error bars indicate mean ± SEM. (G) Representative histogram plots showing the increased expression of CD47 and CD31 on Sparc-/- PMN compared with the WT counterpart (pristane treated day 130). (H) Mean fluorescence intensity (MFI) of expression of CD47 and the relative fold reduction in PMN isolated at day 130 after pristane treatment and following o/n treatment with the NF-?B activation inhibitor InSolution (n = 6/group). Statistical analysis: CD47 MFI, one-way ANOVA, multiple comparison test; fold of reduction: Student's t test (*p < 0.05, ****p < 0.0001). Error bars are mean ± SEM. (I) Phosphorylation of p65 and of Ikba in WT and Sparc-/- PMN treated for 2 h with LPS (n = 6/group). Data are expressed as ratio between phospho-p65/p65 and phospho IkBa/IkBa. Statistical analysis: one-way ANOVA, multiple comparison test *p < 0.05; **p < 0.01; ***p < 0.001 (Tukey). Error bars are mean ± SEM.