Rcn 2

Human Cardiac Fibroblasts were obtained from Promocell and maintained in medium Fibroblasts Media 3. Cells were cultured according to the manufacturer´s instructions. Cells were used between passages 5–7. Cells were seeded into six-well plates at 90% confluence and serum starved for 12 h and then stimulated with Aldosterone (10⁻⁸ M, Sigma), Gal-3 (10⁻⁸ M, R&D Systems), CT-1 (10⁻⁷ M, R&D Systems), RCN-1 (0.01, 0.1 and 10 µg/mL, Abcam), RCN-2 (0.01, 0.1 and 10 µg/mL, Abcam), RCN-3 (0.01, 0.1 and 10 µg/mL, Abcam), Spironolactone (Spiro, 10⁻⁶M, Sigma) and Angiotensin II (Ang II, 10⁻⁹–10⁻⁷M, Sigma) for 24 hours for protein analysis. Aldosterone was prepared in ethanol at 10⁻²M and then diluted in culture medium to be used at 10⁻⁸M. Recombinant Gal-3 and CT-1 were reconstituted in PBS. The doses were chosen based on preliminary and previous studies^{6 (link),22 (link),30} .
For the intracellular pathways study, cells were treated with RCN-3 for 5, 15, 30 and 60 minutes. The following chemical inhibitors were added at 10⁻⁵ mol/L 1 hour prior to RCN-3 stimulation: Wortmannin (Sigma Aldrich), PD98059 (Sigma Aldrich) and AG-490 (Sigma Aldrich).

Total proteins from human cardiac fibroblast were separated by SDS-PAGED on 10% polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Membranes were probed with primary antibodies for RCN-1 (Abcam; dilution 1:500), RCN-2 (Abcam; dilution 1:500), RCN-3 (Abcam; dilution 1:500), collagen I (Sigma; dilution 1:500), collagen III (Santa Cruz; dilution 1:500), Fibronectin (Millipore; dilution 1:1000), Gal-3 (ThermoFisher; dilution 1:1000), CT-1 (Abcam; dilution 1:500), connective tissue growth factor (CTGF; Torrey Pines Biolabs Inc., dilution 1/1000), ERK1/2 and ERK1/2-P (Thr202/Tyr204) at 1/1000 (Cell Signaling), Akt and Akt-P (Ser473) at 1/1500 (Cell Signaling), Stat3 and Stat3-P (Tyr705) at 1/1500 (Cell Signaling). Western blots were performed with stain-free gels for loading control. After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham). After densitometric analyses, optical density values were expressed as arbitrary units. Results are expressed as an n-fold increase over the values of the control group in densitometric arbitrary units. All Western Blots were performed at least in triplicate for each experimental condition.