Figure 4
Effects of angiotensin II type 2 receptor (AT2R) gene silencing and AT2R activation with the agonist CGP42112A on the expression of pro-inflammatory cytokines in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). (a) Verification of silencing capacity of AT2R small interfering RNA (siRNA). Protein levels of AT2R in RA-FLS transfected with AT2R siRNA or non-silencing scrambled RNA (SCR) were measured by Western blotting; α-tubulin was used as a loading control. Representative cropped immunoblots are shown. All samples were run on the same gel. Numbers on the right indicate molecular weight (kDa). (b) Gene expression of IL1B, IL6 and TNF was evaluated by Real-Time PCR in RA-FLS at baseline, after transfection with AT2R siRNA or non-silencing SCR, and after treatment with AT2R agonist. The mRNA expression levels were normalized to the 18 S ribosomal RNA gene. For each gene, the mRNA expression in basal cells was set to 1; the other results are expressed as x-fold increase/decrease over basal response. Bars represent the mean ± SEM. Results are representative of three independent experiments performed with each one of the six RA-FLS lines. Statistical analysis was carried out with Student’s t test; *p < 0.05, **p < 0.001 versus the respective basal condition.
