Pd123319

Ang II, ET-1, PD123319, gallein (G_βγ inhibitor), and BQ788 were obtained from Tocris Bioscience (Ellisville, MO, USA). Recombinant human TGF-β1, valsartan, bosentan, ambrisentan, LY2109761, FR180204, and SB203580 were obtained from Sigma Aldrich (Saint Louis, MO, USA). SIS3 (Smad3 inhibitor) and FR900359 (G_αq inhibitor) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Fibroblast growth medium and related cell culture reagents were obtained from Promocell (Heidelberg, Germany).

The following drugs were used: pentobarbital purchased from Serva (Heidelberg, Germany); inactin, losartan, and L-NAME from Sigma-Aldrich (St. Louis, MO, USA); DAA-I and Ang II from Bachem (Bubendorf, Switzerland); and PD123319 (Tocris, Minneapolis, MN, USA).

Angiotensin II (Ang II) was purchased from Sigma or Bachem. Due to variation in potency of various batches of Ang II, this reagent was used at 10⁻⁶ M, as this concentration gave consistent results with all batches. Losartan (Sigma), PD123319 (Tocris BioScience), and U0126 (Millipore) were used at 10⁻⁵ M each, while matrix metalloprotease 2 (MMP-2) inhibitor-1 (MMP2 I1, cis-9-Octadecenoyl-N-hydroxylamide, Oleoyl-N-hydroxylamide, OA-Hy; Millipore) was used at 3×10^-5 M. Protease (PIC) and phosphatase (PPIC II and PPIC IV) inhibitor cocktails, were from Sigma and Calbiochem, respectively.

To determine the potential signaling mechanisms involved in Ad-Sglt2-ECFP/Ang II-induced biological responses, WT and Agtr1a^-/- mPCT cells expressing Ad-Sglt2-ECFP/Ang II were concurrently treated with the AT₁ receptor antagonist losartan (10 µM; Tocris, Minneapolis, MN, USA), the AT₂ receptor antagonist PD 123319 (10 µM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 µM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 µM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 µM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 µM; MCE, Belleville, NJ, USA).

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.

We purchased CNQX, MK-801, Ang-II, losartan, and picrotoxin from Sigma and TTX and PD123319 from Tocris Biosciences. Drugs were prepared immediately before use in either distilled water (Ang-II, TTX, PD123319, and losartan) or DMSO (CNQX, MK-801, and picrotoxin) and diluted in aCSF to achieve the desired concentration. We used 500 nm Ang-II for our experiments, a high working concentration for a ligand-receptor pair with a K_d < 5 nm. However, we used a high Ang-II concentration to ensure adequate slice penetration of bath-applied Ang-II.