Hif142

We used one slow growth, androgen-sensitive prostate cancer cell line LNCaP (DSMZ, Braunschweig, Germany) and invasiveness, androgen-insensitive cell line PC3 (ECACC). In 2014, the lines were authenticated by short-tandem repeat (STR) DNA profiling (LGC Standards Cell Line Authentication Service, Germany). Human prostate cancer cells were maintained as a traditional monolayer culture in RPMI (Gibco, Thermo Fisher Scientific Inc, Waltham, MA, USA) with 10% heat-inactivated fetal bovine serum (FBS) and standard supplements, such as: sodium pyruvate, L-glutamine, HEPES buffer and antibiotics (PSN). Ang-(1-9) (no. H-5038) and Ang-(3-7) (no. H-6965) were purchased from Bachem (Bubendorf, Switzerland), while angiotensin receptor inhibitors were purchased from TOCRIS (Bristol, UK): losartan (AT1 antagonist, no. 3798), PD123319 (AT2 antagonist, no. 1361), A779 (AT1-7/MAS antagonist, no. 5937), HIF142 (AT4/IRAP antagonist, no. 5627). The final concentrations of both angiotensins were 1nM whereas antagonists were used at 1000 nM. Given that these peptides are relatively quickly degraded, the experimental medium was changed every 24 h.

Ang1–7 (H-1715) was purchased from Bachem. The angiotensin receptor blockers losartan (AT1 antagonist), PD 123319 (AT2 antagonist), A779 (AT1–7/MAS antagonist) and HIF142 (AT4/IRAP antagonist) were obtained from TOCRIS Bioscience. For all experiments, heptapeptide was used at a final concentration of 1 nM, and inhibitors at a concentration of 1000 nM. This concentration was selected on the basis of earlier research work and literature data. Medium containing the mentioned above compounds was changed every 24 h. Unless otherwise specified, the medium and other culture supplements were purchased from Gibco; Thermo Fisher Scientific, Inc.