Brp lpa

Anti–PD-1 and anti–PD-L1 antibodies were purchased from Bio X Cell (clone RMP1-14 and clone 10F.9G2, respectively), and treatments were given once weekly via i.p. injection at 200 μg/treatment in 100 μL total volume. PF-8380 was purchased from Selleckchem and dissolved in 1% CMC-Na plus 0.9% NaCl. Mice were dosed orally daily at 50 mg/kg in 100 μL. BrP-LPA was purchased from Echelon Biosciences and was dissolved in sterile 0.9% saline. Treatments were given at 1 mg/mL twice weekly via 100 μL i.p. injection. AS2717638 was purchased from MedChemExpress and was dissolved in the following: 10% DMSO plus 40% PEG300 plus 5% Tween-80 plus 45% saline. Mice were treated orally daily at 10 mg/kg in 100 μL. For syngeneic tumor experiments, treatments were started after palpable tumor formation at 1 week after implantation. For GEMM treatments, once lung tumors were confirmed by CT scanning, mice were randomly enrolled into treatment arms and treated for 4 weeks. The pretreatment CT image was used for baseline measurements, and the week 4 image was used as the endpoint measurement. The percentage difference between these time points was calculated for each mouse. In vitro drug treatment information can be found in Supplemental Methods.

UCB CD34⁺ cells were suspended in X-Vivo medium, i.e., 2 × 10⁵ cells per well of a 96-well plate containing 200 μL of X-Vivo medium supplemented with one of the following antagonists: MK2206 (3 μM, Selleckchem), PD98059 (60 μM, CaymanChem, Michigan, USA), GW9662 (50 μM, Calbiochem), Y-2762/ROCK (50 μM, Calbiochem), Ki16425 (10 μM, Selleckchem) or 1-Bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate (BrP-LPA) (1.5 μM, Echelon Biosciences Inc.). After 10 min of incubation, LPA 100 μM (in 0.01% DMSO X-Vivo) was added and the cells incubated in normoxia (21% O₂, 5% CO₂, 37 °C) conditions for 1 h. Then, the plates were incubated in hypoxic (0.5% O₂, 5% CO₂, 37 °C) conditions for 24 h. Antagonists and LPA were maintained in the culture medium during the 24 h period. Cell survival was determined by annexin V/propidium iodide (PI) as described before.