Figure 3
Expression of angiotensin II type 2 receptor (AT2R) in fibroblast-like synoviocytes (FLS) from healthy, osteoarthritis (OA) and rheumatoid arthritis (RA) synovium at baseline and after treatment with tumor necrosis factor (TNF)-α and interleukin (IL)-1β, alone or in combination. (a) Representative microphotographs of FLS isolated from healthy (H-FLS), OA (OA-FLS) and RA (RA-FLS) synovium subjected to immunofluorescence staining for AT2R (green) and nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars are indicated in each panel. (b) Western blotting of total protein extracts from H-FLS, OA-FLS and RA-FLS at basal condition and treated with TNF-α and IL-1β, alone or in combination, assayed with anti-AT2R antibody; α-tubulin was used as a loading control. Representative cropped immunoblots are shown. All samples were run on the same gel. Non-adjacent samples were separated by a black line. Numbers on the right indicate molecular weight (kDa). A protein band with the expected molecular weight of 41 kDa was detected with the anti-AT2R antibody. The densitometric analysis of the bands normalized to α-tubulin is reported in the histograms. Data are mean ± SEM of optical density in arbitrary units (a.u.). Student’s t test was used for statistical analysis; *p < 0.05 versus the respective basal condition for each cell type. Results are representative of three independent experiments performed with each one of the six H-FLS, six OA-FLS and six RA-FLS lines.
