Fig. 4: CD62L⁺ KCs induce NETosis by CCL8.

a NETosis of murine primary neutrophils cultured with CM of untreated KCs, KCs pretreated with LvM16 CM, or CM of the pretreated KCs, or CM of LvM16 (zoomed areas shown at bottom). b NETosis of murine primary neutrophils cultured in CM of total KCs or CD62L⁺ KCs. c Expression heatmap of secreted protein-encoding genes in KC subclusters. d NETosis of neutrophils cultured with recombinant CCL8 (1 ng/mL), SAA1 or SAA3 (10 ng/mL) or PMA (20 nM) for 16 h. e mRNA and protein expression of Ccl8 in CD62L⁺ and CD62L^– KCs isolated from LvM16 liver metastases of mice. Relative quantitation of blot intensity normalized to loading control was provided below each blot. f NETosis of neutrophils cultured with CM of KCs from WT or Ccl8^–/– mice. The KCs were pretreated with CM of LvM16. g, h ERK phosphorylation and NETosis quantitation by IF analysis of murine neutrophils treated with PMA, recombinant CCL8 (10 ng/mL) and/or the ERK inhibitor SCH772984 (SCH, 1 μM) for 16 h. i ERK phosphorylation and NETosis of neutrophils treated with CCL8 (10 ng/mL) and/or the CCR1 antagonist BX471 (1 nM) for 16 h. j GO analyses of the upregulated genes in rCCL8-treated vs untreated neutrophils. n = 3 (a, e, h, i), 4 (b, d, f) biological repeats per group. Scale bars, 50 μm. P values were obtained by two-tailed unpaired t-test. Data are shown as mean ± SD.