Figure 5

Macrophages play a pivotal role in LPS-induced differentiation by secreting IL-1β. (a) IL-1β concentration in macrophages or myometrial cells stimulation supernatants. Macrophages or myocytes were cultured for 24 h in presence or absence of LPS (100 ng/ml). Supernatants were collected and IL-1β concentrations were evaluated with ELISA assay and standardized with protein cell content ± SEM, n = 6, *** P < 0.001. (b) Schematic representation of the effects of IL-1β blockage for each cell type on differentiation of myometrial cells in a Transwell assay. (c) Transwell experiments with macrophages (upper compartment) and myometrial cells (lower compartment) following treatment with LPS (100 ng/ml) in presence or absence of Anakinra (1 µg/ml) or αIL-1β (100 ng/ml). Representative images of phalloidin (green), vinculin (purple) and DAPI for nuclear localization (blue) were taken with an Axiozoom epifluorescence microscope (×400) in five random chosen fields of four separated experiments. (d) Mean fluorescence intensity of phalloidin or vinculin ± SEM, n = 20, ****P < 0.0001. (e) Fluorescence images of LY (green) and RD (red) transfer to adjacent cells after scrap loading. Cells were treated for 24 h with LPS in the upper compartment and with Anakinra or αIL-1β in the lower compartment. Pictures were taken with an inverted microscope at ×100 magnification in two random chosen fields and are representative of three experiments. (f) Mean surface of LY staining ± SEM, n = 6, ****P < 0.0001.