Fig. 8

The paracrine action of the IL-1β/IL1R1 axis promotes the actin-myosin contractility and the invasion of CAFs. a-b Immunoblots of IL-1β and COX2 in CAFs exposed to conditioned medium (CM) collected from MDA-MB-231 cells cultured upon normoxia or hypoxia (2% O2) (a) and in CAFs treated with vehicle or IL-1β (b), in the presence or absence of IL1R1a. c-d Phosphorylation of MLC in CAFs cultured with conditioned medium (CM) collected from MDA-MB-231 cells exposed to normoxia and hypoxia (c) or IL-1β (d), in the presence or absence of the IL1R1 antagonist IL1R1a. Nuclei were stained by DAPI (blue signal). e-f CAFs exposed to conditioned medium (CM) from MDA-MB-231 cells grown upon normoxia and hypoxia (e) or treated with IL-1β (f), in the presence or absence of the IL1R1 antagonist IL1R1a were stained with FITC-phalloidin to detect F-actin stress fibers (green) and with DAPI to detect nuclei (blue). Fluorescence intensities of pMLC and the number of stress fibers/cell was quantified based on F-actin staining in 20 random fields for each condition; results are expressed as fold change of relative fluorescence units (RFU). Enlarged details are shown in the separate boxes. Scale bar 100 μM. g-h Transwell Matrigel invasion assay in CAFs exposed to conditioned medium (CM) collected from MDA-MB-231 cells grown upon normoxia or hypoxia (g) or treated with IL-1β (h), in the presence or absence of IL1R1a. Cells were counted in at least 10 random fields in three independent experiments performed in triplicate, as quantified in side panels. Scale bar 200 μM. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) p < 0.05