Ki16425

A68930 hydrochloride, (R)-propylnorapomorphine hydrochloride, and cabergoline were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). D-APV, FK506 monohydrate, actinomycin D, L-(−)-norepinephrine (+)-bitartrate salt monohydrate, serotonin, nicardipine, U0126, KN93, oleoyl-L-α-lysophosphatidic acid sodium salt, and Ki 16425 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neurotransmitter libraries (dopaminergic, adrenergic, serotonergic, cholinergic, histaminergic, metabotropic glutamatergic, and GABAergic) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Ginsenoside kit (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg2), used in Fig. 5a, was purchased from Extrasynthese (Genay Cedex, France). Ginsenoside Rb1, Rc, Rd, Re, and Rg1, used in Fig. 5d, were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Herbal extract and herbal medicine-derived compound libraries were kindly donated by the Institute of Natural Medicine, University of Toyama (Toyama, Japan). Each herbal extract used in this study was obtained using a standard method described as follows: each herbal medicine (purchased from Tochimoto Tenkaido (Osaka, Japan)) was extracted in water (10-times volume of herbal medicine) at 100 °C for 50 min, evaporated under reduced pressure, and freeze‐dried to obtain a powder extract. Each herbal extract was redissolved in water.

Human glioblastoma-derived cell lines U251 and LN229 (American Type Culture Collection, Manassas, VA, USA (ATCC)) were grown in 10-cm dishes and maintained in DMEM medium (In Vitro, CDMX, Mexico), supplemented with 10% fetal bovine serum at 37 °C under a 95% air, 5% CO2 atmosphere. 1-Oleoyl Lysophosphatidic Acid (LPA; 62215; Cayman Chemical, Ann Arbor, MI, USA) was used to activate LPA receptors and subsequently PKCα. LPA_1/3 receptor antagonist Ki16425 (SML0971; Sigma-Aldrich, St. Louis, MO, USA) was added 30 min before the LPA treatment when used. Progesterone (P4) (SML0971; Sigma-Aldrich, St. Louis, MO, USA) was used to activate PR; selective PR modulator, RU486 (M8046; Sigma-Aldrich, St. Louis, MO, USA), was added 30 min before the P4 treatment when used.
The National Institute of Genomic Medicine in Mexico City did proof of cell validation for the U251 cell line and for LN229, we get the ATCC certificate analysis.

For the measurement of cytosolic free calcium levels, 20,000 cells were seeded into 96-well cell culture plates 2 days before measurement. Then, they were washed twice with Hank’s balanced salt solution (HBSS) containing 0.2% BSA and 10 mM HEPES (Sigma-Aldrich). Cells were incubated with 10 μM Fura-2 AM in HBSS containing BSA, HEPES and 0,25% pluronic acid (Sigma-Aldrich) for 60 min at 37 °C, were washed twice and 100 μl HBSS+HEPES+BSA were added to each well. Analyses were performed in a microplate fluorometer with integrated pipetting system (BMG Labtech NOVOstar, Offenburg, Germany) at 340 nm resp. 380 nm for excitation and 510 nm for emission. Cells were allowed to adapt to 37 °C for 10 min before LPA 18:1 (Avanti Polar Lipids Inc., Alabaster, AL, USA), solved in PBS containing 0.2% human serum albumin in indicated concentrations, was added. In cell line experiments, Ki16425 (Sigma-Aldrich) was used as LPA receptor antagonist in concentrations of 0, 2, or 20 μM 1 min prior to the stimulation with LPA. The change in cytosolic free calcium ([Ca²⁺]_i) was calculated by dividing the measured fluorescence intensity at 510 nm with different excitation wavelengths (ratio 340 nm/380 nm). The baseline levels were subtracted. Adenosine triphosphate was used as a positive control, PBS with 0.2% human serum albumin as a negative control.

LPA (Oleoyl-L-α-lysophosphatidic acid sodium salt, L-7260) and ki16425 (SML0971) were purchased from Sigma (St. Louis, MO, USA).

Whole-embryo culture was performed as described previously (Koike et al., 2009 (link)). In brief, embryos were dissected at E7.5 and cultured in 100% rat serum (Charles River) supplemented with 2 mg/ml glucose in a culture bottle placed in a rotation drum culture system (Ikemoto Rika) at 37°C under 5% O₂, 5% CO₂, and 90% N₂ for 24 h. In the pharmacologic experiments, embryos were incubated with the following reagents: 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl]benzylsulfanyl) propanoic acid (Ki16425, Sigma; 10 µM), (R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester (VPC23019, Avanti Polar Lipid; 10 µM), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolynyl)sulfonyl] homopiperazine dihydrochloride (H1152, Calbiochem; 0.1 mM), phenylarsine oxide (Sigma-Aldrich; 0.1 µM), monensin (Nacalai Tesque; 50 nM), leupeptin (Peptide Institute; 25 µg/ml), 5-(N-ethyl-N- isopropyl)-amiloride (EIPA, MedChemExpress; 50 µM), dynasore (Tocris Bioscience; 20 µM), and pitstop 2 (Selleck Chemicals, 10 µM).