Fig. 4
Hp OMVs induce a reactive astrocyte phenotype in vitro. a Scheme of various treatments. Primary astrocyte cultures were left untreated (control) or treated with 2.5 µg/ml of OMVs for 12 h or with TNF (10 ng/ml for 48 h, positive control). b–e The data correspond to the cell lysates from untreated astrocytes (Ctrl), treated with TNF, or with OMVs. Immunoblot analysis of β3 integrin (b), vimentin (c), GFAP (d), and connexin 43 (e), where β-actin is the loading control. Values in the graphs are means ± S.E.M. of the ratio between the densitometric value of the first antibody signal and that of the respective β-actin (n = 3). *p < 0.05, **p < 0.01. All bands shown in each panel are from the same blot. The vertical line between bands indicates a picture cut. f Confocal microscopy microphotographs of the subcellular localization of connexin 43 (Cx43, middle panel) in untreated astrocytes (control) or treated with Hp OMVs, which are stained for nuclei (blue), Cx43 (green), and actin cytoskeleton (red) (scale bar = 10 µm). g Astrocyte migration in the wound closure assay at 0 and 24 h post-scratch. Cells were pretreated or not with OMVs (2.5 µg/ml for 12 h), and then stimulated with Thy-1-Fc/protein-A (4 µg/ 0.4 µg per well) for 24 h, and cells treated with 3% FBS are the positive controls. Dashed yellow lines indicate the wound border. h Values in the graphs are the means ± S.E.M. of the % of wound closure in Control, TNF- or OMV-treated astrocytes stimulated or not with SFM (control), TRAIL-R2-Fc (negative control), or Thy-1-Fc. The positive control was 3% FBS (n = 3). ns, non-significant, *p < 0.05, and **p < 0.01