Pictilisib

All small molecule inhibitors were obtained from Selleck Chemicals (Houston, TX, USA), these inhibitors include: Sorafenib tosylate (#S1040), PI3K inhibitors: LY294002 (#S1105), Buparlisib (#S2247), Pictilisib (#S1065), Alpelisib (#S2814) and Akt inhibitors MK2206 (#S1078), Ipatasertib (#S2808), Afuresertib (#S7521). All chemicals formulated by their suppliers’ recommendations.

AT9283, AZD4547, AZD6244, BGJ398, crizotinib, pictilisib, ibrutinib, PD173074, ruxolitinib, TAE684, and gandotinib were all purchased from Selleck Chemicals, AUY922, dovitinib, everolimus, gefitinib, mubritinib, saracatinib, sunitinib, and ZSTK474 were from LC Laboratories, CH5424802 was from Active Biochem, SB525334 was from Tocris, and fedratinib was from Axon Medchem. The human gastric cancer cell line SNU-16 was obtained from ATCC and was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum according to the manufacturer’s instructions.

All primary antibodies used are listed in Supplementary Table 2. Cy3- and horseradish peroxidase-conjugated secondary antibodies were all from Jackson Immunochemicals (LABNET OY, Helsinki, Finland) and Alexa488-conjugated secondary antibodies were from Invitrogen. TRITC-Phalloidin and 2-(4-amidinophenyl)-1H -indole-6-carboxamidine were purchased from Sigma-Aldrich (Merck, Helsinki, Finland). Hygromycin B and Puromycin were from Thermo Fisher Scientific (Helsinki, Finland). dimethyl sulfoxide was from Sigma-Aldrich. The MEK inhibitor PD98059 was from LC Laboratories (Woburn, MA, USA). The PI (3) K inhibitor Pictilisib was from Selleck Chemicals (SMS Gruppen, Rungsted, Denmark).

The precursor pictilisib (Figure 2) was purchased from Selleck Chemicals (Houston, TX, USA). Acetonitrile, ethyl alcohol, and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). The water used refers to ultrapure water. The filters used to sterilize the PET tracer solution were Millex-GP 0.22 µm (Millipore, Inc., Bedford, MA, USA). A reference standard [¹²C]-pictilisib was synthesized. Radiolabeling of [¹¹C]-pictilisib was performed with a radiosynthesis module (GE TraceLab FXC-Pro®; GE Healthcare, Brussels, Belgium). The reaction was stirred and heated to 65°C for 5 min and then cooled to room temperature quickly using liquid nitrogen. The reaction mixture was purified by semipreparative high-performance liquid chromatography (HPLC) (column: Phenomenex Luna 5 µm C18 (2) 100A; 250 × 10 mm; mobile phase: EtOH/H₂O: 43/57; flow rate: 2.8 ml/min; λ = 254 nm). In short, the mixture was diluted with 600 μL of 43% ethanol and then injected into the HPLC loop. The product was passed through a sterile 0.22 µm filter and collected into a sterile vial. Subsequently, the product was analyzed through analytical HPLC (column: Phenomenex Luna 5 µm C18(2) 100A; 250 × 10 mm; mobile phase: EtOH/H₂O: 43/57; flow rate: 2.8 ml/min) by coinjecting with standard methyl-pictilisib into the analytical HPLC.

Human embryonic kidney (HEK) 293 cells were obtained from American Type Culture Collection (ATCC, Manassas) and maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Rat PASMC (RPASMC) was isolated and cultured in DMEM supplemented with 10% FBS as reported previously (Zeng et al. 2015).
RPASMCs were starved for 12 h with DMEM supplemented with 0.2% FBS for 12 h and treated with cytokines and growth factors (ANGII [200 ng/mL], ET‐1 [25 ng/mL], FGF2 [30 ng/mL], IGF1 [30 ng/mL], PDGF‐AA [30 ng/mL], PDGF‐BB [30 ng/mL], TGF‐β [20 ng/mL], VEGF [30 ng/mL], and FBS [10%], R&D) for specific time periods (0 h, 2 h, 4 h, 6 h, 12 h, 24 h). For kinase inhibition studies, before being stimulated with growth factors, starved cells were pretreated with corresponding inhibitors (imatinib [5 μmol/L], U0126 [10 μmol/L], SH‐4‐54 [5 μmol/L], enzastaurin [5 μmol/L], pictilisib [1 μmol/L], Selleck) for 0.5 h.