Fig. 5
Hp OMVs-enhanced NF-κB nuclear translocation and activity are required for β3 integrin and connexin 43 upregulation in astrocytes. a, b Immunoblots of p65 NF-κB (a), (pS536p65) NF-κB (b) and β-actin (loading control) of astrocytes treated for 12 h with 2.5 µg/ml OMVs, with or without 1 µM IMD 0354 or 1 µM BMS 344551. Astrocytes incubated with TNF (10 ng/ml, 48 h) are the positive control samples. c Confocal microscopy of nuclear p65-positive cells (red), nuclear protein lamin-associated with peptide 2 (LAP-2) (green), and DAPI (blue) in astrocytes treated for 60 min with OMVs in the presence or absence of 1 μM IMD 0354 or 1 µM BMS 344551 (scale bar = 10 µm). The right panel is the merged image of LAP2, p65 and DAPI (scale bar = 10 µm). White and yellow arrows indicate the absence or presence of NF-κB in the nucleus, respectively. d Values in the graph represent the percentage of nuclear p65-positive cells (red nuclei) from the total nuclei (DAPI/LAP-2) (mean ± S.E.M.). The average number of cells was 100 astrocytes per condition, per experiment (n = 3). e ELISA assay of NF-κB DNA-binding activity in equal amounts of nuclear extracts obtained from astrocyte cultures treated for 60 min as indicated with Hp OMVs or TNF, with or without 1 μM IMD 0354 or 1 μM BMS 344551. f, g Immunoblots of connexin 43 (f), β3 integrin (g), and β-actin (loading control) of astrocytes treated with 2.5 µg/ml of Hp OMVs for 12 h, or with TNF (10 ng/ml, 48 h), ± 1 µM IMD 0354 or 1 μM BMS 344551. In all graphs, values are means ± S.E.M.; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared to control; #p < 0.05, ##p < 0.01, compared to TNF; and †p < 0.05, ††p < 0.01, †††p < 0.001, compared to OMVs