Figure 4.

The adenosine metabolite, inosine (100 µM), mimicked the inhibitory effect of the adenosine A₃ receptor agonist, 2Cl-IB-MECA (100 nM), on cells proliferation/viability (MTT assay, A and C) and collagen production (Sirius Red staining, B and D) by human subcutaneous fibroblasts (HSCF) grown in culture for 28 days. Inhibition of HSCF cells proliferation by 2Cl-IB-MECA (100 nM, A) and inosine (100 µM, C) was attenuated by MRS1191 (10 nM), but the selective A₃ receptor antagonist did not affect the inhibitory role of 2Cl-IB-MECA (100 nM, B) and inosine (100 µM, D) on collagen production. Boxes and whiskers represent pooled data from at least three different individuals; 4–6 replicas were performed for each individual. * p < 0.05, ** p < 0.01 and *** p < 0.001 (two-way ANOVA) represent significant differences compared to control conditions; ^# p < 0.05 and ^### p < 0.001 (two-way ANOVA) represent significant differences compared to the effect of 2Cl-IB-MECA in the same set of experiments. Panel E shows that A₃ receptor expression (green) was kept fairly constant with time of the cells in culture, as evidenced both by immunofluorescence confocal microscopy and by Western blot analysis normalized either by β-actin (left hand-side blots) or β-tubulin (right hand-side blots) content of the cells. Nuclei are stained in blue with DAPI. Shown are representative data of cells from four different individuals cultured for 7 and 28 days, respectively; experiments were performed in triplicate.