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

Fig. 4. Nicotine polarizes N2-neutrophil promotes cancer cell stemness by upregulating exosomal miR-4466.

Fig. 4

A Experimental setup for obtaining control or nicotine-activated neutrophil CM or exosome from primary neutrophils (human, mouse). H2030BrM cells were treated with indicated CM or exosomes (equivalent to 10 µg of protein) overnight followed by examining the CSCs population (CD44+ESA+) by FACS (n = 3/independent experiment, unpaired two-tailed t-test). B H2030BrM cells were treated with indicated exosomes overnight and examined for CSCs by FACS (left panel), primary and secondary sphere formation (right panel), SOX2, OCT4, and NANOG expression by qRT-PCR and immunoblotted for SOX2 and NANOG (bottom panels, n = 3/independent experiment, unpaired two-tailed t-test). C miRNA sequencing analysis was performed on exosomes from control or nicotine-treated neutrophils (human primary (n = 3), FC > 5, p < 0.05)). D Expression of serum/urine-derived exosomal miR-4466 in cancer-free subjects with smoking history. E Strategy to identify miRNA target using previously published dataset (GSE14108) [46] in combination with TFcheckpoint database. F H2030BrM cells transduced with control or miR-4466 lentiviral plasmid and examined for SKI and SOX2 expression by qRT-PCR and immunoblotted for SOX2 (n = 3/independent experiment, unpaired two-tailed t-test). G H2030BrM cells were treated with indicated exosomes or transfected with SKI 3′-UTR luciferase reporter plasmid with indicated miRNAs along with phRG-TK Renilla luciferase plasmid (internal control). Luciferase activities were measured at 24 h post-transfection (n = 3/independent experiment, unpaired two-tailed t-test). H SOX2 promoter activity was measured in 293T cells that were co-transfected with SKI-expressing plasmid and phRG-TK Renilla luciferase plasmid (internal control). Values were expressed as the normalized ratio of firefly to Renilla luciferase (n = 3/independent experiment, unpaired two-tailed t-test). β-Actin and GAPDH were used as a normalization controls. All experiments were repeated three times independently, and each experiment showed similar results. Data are presented as mean ± S.E.M.