1 oleoyl lysophosphatidic acid lpa

RhoA G-LISA (Cytoskeleton, Inc) was performed according to the manufacture's specifications. Briefly, monolayer cells were grown to confluence before being stimulated with 10 µM 1-Oleoyl Lysophosphatidic acid LPA (Cayman Chemical) for 5 minutes. Cells were collected using the lysis buffer from the kit. A minimum of 200 spheroids were collected at 48, 72, 96, and 120hr. Spheroids were collected on ice, centrifuged briefly to pellet. Lysis buffer was added and the spheroids were dissociated before starting the assay. Each condition was testing in triplicate and the experiment was performed thrice. Standard deviations (SD) were expressed from at least triplicate wells for a representative experiment. Luminescence was measured on the SpectraMax5 (Molecular Devices).

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.