Fig. 4: Nicotine-activated N2 neutrophils promote MET phenotype. | Nature Communications

Fig. 4: Nicotine-activated N2 neutrophils promote MET phenotype.

From: RETRACTED ARTICLE: Nicotine promotes breast cancer metastasis by stimulating N2 neutrophils and generating pre-metastatic niche in lung

Fig. 4

A Upper panel: Experimental setup for obtaining control or nicotine-polarized neutrophil conditioned medium. Lower panels: Morphological changes in MDAMB231 and MCF10CA1a cells treated with the indicated CM. B MDAMB231 and MCF10CA1a cancer cells were treated with the indicated nicotine-activated neutrophil CM for 24 h. Cells were then examined for the expression of epithelial (E-cadherin, EpCam, Krt18) and mesenchymal (Vimentin, N-cadherin, ZEB1) markers by qRT-PCR and western blot. β-Actin was used as input control for qRT-PCR. Representative immunoblots shows expression of E-cadherin, Vimentin, and ZEB1 in MDAMB231 and MCF10CA1a. GAPDH was used as a control for western blot [n = 3 individual experiment/group, unpaired two-tailed t-test, ***p = 0.0003, *p = 0.02, *p = 0.01, **p = 0.007 (MDAMB231), ***p = 0.001, ***p = 0.0008, **p = 0.001, ***p = 0.0004 (MCF10CA1a)]. C Representative IHC images for E-cadherin in metastatic lungs derived from the experiment in Figs. 1B and 2A (n = 3/individual experiment; scale bar: 100 µm). D MDAMB231, MCF10CA1a, and MCF7 cancer cells were treated with or without nicotine (1 µM) for 24 h followed by examining the expression of epithelial (E-cadherin, EpCam) and mesenchymal (Vimentin, ZEB1) markers by qRT-PCR. β-Actin was used as a control for normalization [n = 3 individual experiment/group, unpaired two-tailed t-test, *p = 0.03, ****p < 0.0001, **p = 0.003 (MDAMB231), ***p = 0.0003, **p = 0.001, **p = 0.007, *p = 0.03 (MCF10CA1a), **p = 0.004, ***p = 0.0002, **p = 0.001 (MCF7)]. E MDAMB231 cancer cells were treated with either nicotine or with conditioned medium derived from nicotine-treated human primary neutrophils. Cells were then examined for their colony-forming ability (n = 3 individual experiment/group, unpaired two-tailed t-test, **p = 0.003). F Expression of α4 and β2 nicotine receptors in primary neutrophils (human and mouse) and in various breast cancer cells (MCF7, MDAMB231, MCF10CA1a, BT549) was examined by qRT-PCR. β-Actin was used as a control for normalization (n = 3 individual experiment/group). All data are presented as mean ± S.E.M.

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