As605240

The ICCB Known Bioactive Library was purchased from BIOMOL (Enzo Life Sciences) and used for the adult zebrafish transplantation-based chemical screen. Chemicals were diluted at a 1:200 ratio. Chemicals used for the secondary round of screening for confirmation were from a different aliquot of the library, independent of the primary screen plate. 11,12-EET (Cayman Chemical, Cat. 50511) was resuspended in DMSO with original organic solvent evaporated. AS605240 (Sigma-Aldrich Cat. A0233) was resuspended in DMSO. The following chemicals were used for zebrafish marrow treatment: dmPGE2 (Cayman, Cat. 14750), 10 μM; BIO (EMD), 0.5 μM. 0.5 μM 11,12-EET and 14,15-EET were used for zebrafish WKM treatment (Fig. 1e); 2 μM 11,12-EET for all mouse WBM treatment (Fig. 4); 5 μM 11,12-EET for all zebrafish embryo treatment (Fig. 2, 3). The concentrations were chosen based on dose titration pilot experiments with doses spanning 0.1–50 μM. For the chemical suppressor screen, the suppressors were added 30 min prior to 11,12-EET. Zebrafish embryos were incubated with inhibitors at three different concentrations. The highest effective concentrations tested without causing general toxicity are listed in Supplementary Table S1.

Primary murine pulmonary endothelial cells (PMECs) were isolated as we have described previously16 (link). PMEC were re-suspended in diluted ECGM (1:3 in DMEM/F12) and plated on 15 mg/mL reduced-growth factor extracellular matrix (Cultrex, Trevigen, USA) at a density of 1.75 × 10⁵ cells/cm². Tubule formation was determined by measuring branch number and length using NIH Image J software, 2, 4 and 6 h after treatment with CNP (1 nM to 1 μM, GenScript, USA) or VEGF (30 ng/mL, Pre-protech, USA).
Tubule formation in HUVEC was measured following treatment with CNP (1 nM) or ANP (10 nM) in the absence or presence of selective NPR-C antagonist M372049 (10 μM), the selective PI3Kγ inhibitor AS605240 (100 nM; Sigma), or the selective PKG blocker KT5283 (2 μM; Sigma) over a 16 h period. A cohort of cells was transfected using lipofectamine 2000 (Thermofisher Scientific) with either non-specific siRNA (Mission Control, Sigma Aldrich), NPR-B- or NPR-C- specific siRNA (Sigma Aldrich) and were treated with CNP or the selective NPR-C agonist, cANF4 (link)–23 (link) (2 nM). Silencing of NPR-B and NPR-C was confirmed at the mRNA and protein level using RT-PCR and immunoblot (Supplemental Figure 1A-C).

IC-87114 (PI3Kδ inhibitor) was obtained from Selleckchem (Houston, TX; S1268) and reconstituted to maximal solubility of 2.52 mmol/L in DMSO, and aliquots were stored at −80°C. AS-605240 (PI3Kγ inhibitor) was obtained from Sigma-Aldrich (Natick, MA; A0233) and reconstituted to 9.7 mmol/L in DMSO, and aliquots were stored at −80°C. The following inhibitors were obtained from Novartis (Cambridge, MA): BKM-120 (pan PI3K), BYL-719 (PI3Kα inhibitor), and MEK-162 (MEK inhibitor). These were reconstituted in DMSO 10 mmol/L, and aliquots were stored at −20°C.