Kt5823

H₂S production was evaluated according to Stipanuck and Beck [32 (link)] with modifications [33 (link),34 (link)]. Human urothelium samples were incubated with 8-Br-cGMP (100 μM) or d-cAMP (100 μM) for 30 min. T24 cells were incubated with 8-Br-cGMP (100 μM) or d-cAMP (100 μM) for 5, 15 and 30 min. The incubation time of 15 min was chosen as optimal time and used in all successive experiments in T24 or CBSΔT24 cells. The selective inhibitors of PKG (KT5823, 10 μM, Tocris, UK) or PKA (KT5720, 10 μM, Tocris, UK) were added for 20 min prior to challenge with 8-Br-cGMP or d-cAMP, respectively.

T24 cells treated with carbachol (0.1, 1 and 10 μM, for 5 min) were analyzed by western blotting in presence or in absence of KT5823 (10 μM), a PKG inhibitor (Tocris, UK). Protein samples (30 μg) were resolved by 12% SDS-PAGE and analyzed as previously described38 39 (link).The membranes were incubated with rabbit polyclonal anti-CBS, (1:1000 Santa Cruz Biotechnology, Heidelberg, Germany), and anti-pCBS^Ser227 (1:400, PRIMM srl, Milano, Italy)21 . In another setting of experiments T24 cells or human urothelium were blotted for anti-eNOS (1:1000, BD Transduction, CA, USA). The target protein band intensity was normalized over the intensity of the housekeeping β-actin (1:5000, Sigma-Aldrich, Milan, Italy). Data were expressed as mean  ±  SE. Results were analyzed by ANOVA following by Bonferroni as post test. p  <  0.05 was considered significant.

Diethylamine NONOate (NO donor) was purchased from Enzo Life Sciences (Cat # ALX-430-014-M005, Farmingdale, NY, United States). L-NAME (NOS inhibitor, Cat # N5751), Calphostin C (protein kinase C – PKC – inhibitor, Cat # 20785), and ODQ (soluble guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, Cat # O3636) were purchased from Sigma-Aldrich (Saint Louis, MO). A selective protein kinase G (PKG) inhibitor, KT5823, was purchased from TOCRIS (Cat # 1289, Bristol, United Kingdom).
We used a mouse anti-renin polyclonal IgG B-12 antibody (sc-81178) for detection of renin, and ß-actin was detected using a goat anti- ß-actin monoclonal IgG antibody (sc-1615), both purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, United States). For western blot procedures the secondary antibodies used were the IR Dye 800CW anti-goat and mouse according to the primary antibody chosen (Li-Cor Bioscience, NE, United States). For immunofluorescence, we utilized a mouse anti-renin polyclonal IgG H-105 antibody (sc-22752–1:200), and secondary Alexa Fluor antibodies (Alexa fluor-488), both purchased from Life Technologies (Carlsbad, CA, United States). The mouse kidney cortical CD cells (M-1 cell line) were purchased from American Type Culture Collection (Cat: CRL-2038, ATCC, Manassas, VA, United States).

The 18 icariin analogs were synthesized and identified as described previously (16 (link), 17 (link), Figure S1). The compounds were dissolved in DMSO (Sigma-Aldrich, Shanghai, China) and stored at -20°C. Testosterone was purchased from Sigma-Aldrich. The magnetic particle-based 17β-estradiol enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bio-Ekon Biotechnology (Beijing, China). NSC-87877, KT5823, PD98059 and Rp-8-pCPT-cGMPS were obtained from Tocris Bioscience (MN, USA). Antibodies used in this study as follow: aromatase (1:1,000, ab64881, Abcam, Shanghai, China), Src (1:1,000, 11097-1-AP, Proteintech, Wuhan, China), shp2 (1:2,000, 20145-1-AP, Proteintech, Wuhan, China), phospho-Src-Tyr⁴¹⁸ (1:1,000, 11091, SAB, Nanjing, China), phospho-Src-Tyr⁵²⁹ (1:1,000, 11153-1, SAB, Nanjing, China), ERK1/2 (1:1,000, 48504-1, SAB, Nanjing, China), phospho-ERK1/2 (1:1,000, 12082-1, SAB, Nanjing, China) and PDE5A (1:1,000, 37810, SAB, Nanjing, China).

Sodium nitroprusside (SNP) was obtained from Millipore (Billerica, MA, USA) and dissolved in 0.3x Danieau’s solution (19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO₄, 0.2 mM Ca(NO₃)₂, 1.7 mM HEPES, pH 7.2) immediately before use in experiments. KT5823 was obtained from Tocris (Bristol, UK). Stock solutions of KT5823 were prepared by dissolving in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan). L-NAME was obtained from Dojin Chemicals (Kumamoto, Japan). 2-Phenoxyethanol was obtained from Wako Chemicals (Osaka, Japan).