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. 2022 Apr 23;41(22):3079–3092. doi: 10.1038/s41388-022-02322-w

Fig. 3. Nicotine promotes N2-neutrophil polarization in a STAT3 dependent manner.

Fig. 3

A Mouse primary neutrophils were treated with vehicle or nicotine (1 µM) overnight and examined for N1(CD95, Nos2, Arg1), N2(Mrc1, Arg1, Arg2) markers by qRT-PCR (n = 3/independent experiment, unpaired two-tailed t-test). B Flow cytometric quantification of circulating N1(Nos2, CD95), N2(Mrc1, Arg2) neutrophils at day 0 and day 10 in tumor-free mice pretreated with vehicle or nicotine (n = 4 mice/group, randomly selected, unpaired two-tailed t-test). C Representative immunofluorescence staining and quantification (number of cell infiltrated/high power field (HPF)) of N1(NOS2); N2(ARG2) neutrophils in lung cancer brain metastatic tissues of never smoker (n = 6) and current smoker (n = 20) (unpaired two-tailed t-test, Scale bar: 50 µm). D Primary neutrophils (human, mouse) were treated with vehicle or nicotine (1 µM) overnight and examined for STAT3 expression by qRT-PCR (n = 3/independent experiment, unpaired two-tailed t-test). E HL-60 cells were treated with or without STAT3-siRNA in the presence of nicotine (1 µM/48 h) followed by examining N2-neutrophils marker expression by qRT-PCR (n = 3/independent experiment, unpaired two-tailed t-test). F Relative proportion of mature (segmented) and immature (ring-shaped) neutrophil populations isolated from mice peripheral blood as assessed by hematoxylin & eosin staining (used in A, Fig. 2; unpaired two-tailed t-test). G Cells in F were examined for c-kit expression by qRT-PCR (n = 3/independent experiment, unpaired two-tailed t-test). β-Actin were used as normalization controls. All experiments were repeated three times independently, and each experiment showed similar results. Data are presented as mean ± S.E.M.