Fig. 4

Fn-induced NETs facilitate metastasis of CRC in vitro. A The procedure of transwell migration and invasion assay for CRC cells co-cultured with (Neu), (Neu + Fn), (Neu + Fn + DNase I) and (Neu + PMA). B-D Transwell migration assay for CRC cells co-cultured with (Neu), (Neu + Fn), (Neu + Fn + DNase I) and (Neu + PMA) (B). Quantification analysis of the number of transmembrane cells (C, D). E Western blot analysis of E-cadherin, N-cadherin and vimentin in CRC cells treated with (Neu)-CM, (Neu + Fn)-CM, (Neu + Fn + DNase I)-CM for 48 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. F–H Transwell migration assay for CRC cells treated with or without NETs (F). Quantification analysis of the number of transmembrane cells (G-H). I Western blot analysis of E-cadherin, N-cadherin and vimentin in CRC cells treated with or without NETs. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. J-L Transwell invasion assay for CRC cells co-cultured with (Neu), (Neu + Fn), (Neu + Fn + DNase I), (Neu + PMA) (J). Quantification analysis of the number of transmembrane cells (K-L). M Western blot analysis of MMP9 and MMP2 in CRC cells treated with (Neu)-CM, (Neu + Fn)-CM, (Neu + Fn + DNase I)-CM for 48 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. N-P Transwell invasion assay for CRC cells treated with or without NETs (N). Quantification analysis of the number of transmembrane cells (O-P). Q Western blot analysis of MMP9 and MMP2 in CRC cells treated with or without NETs. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. R-U Adhesion assay for Dil-labeled CRC cells trapped within Fn-induced NETs in vitro. Representative fluorescence images were shown. White scale bars: 50 µm. The number of Dil-labeled CRC cells is quantified and shown in the right panel, respectively. White scale bars: 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001