Jph203

CCRF-CEM and MDA MB231 cells were treated with FK866, CHS-828 (200484-11-3, Cayman Chemical), oligomycin A (75351, Sigma), 2-deoxyglucose (D8375, Sigma), GSK2837808A (GSK) (5189, Tocris), JPH203 (a selective L-type amino acid transporter), and l-asparaginase (11185, Sigma) for 48 h. JPH203 was kindly obtained from Dr. Peyron [30 (link)]. In vitro drug sensitivity was assessed by the colorimetric methyl-thiazolyltetrazolium (MTT) assay (sigma), XTT proliferation kit (sigma), and OZBlue Cell Viability kit (OZbiosciences). Combination treatment of FK866 with JPH203 was performed and 48 h after combination treatment, Cell viability was determined using XTT assay (Invitrogen) and DAPI staining. Percentage of cell death was subjected for drug combination analysis as described by combination index (CI). CI was analyzed using CompuSyn software V1.0 by the method of Chou and Talalay [31 (link)]. CI < 1 indicates drug synergistic effect, CI > 1 indicates drug antagonistic effect, and CI = 1 indicates drug additive effect.

A total of 4 × 10⁶ neutrophils were plated in quadruplicate into a Seahorse XF96 Cell Culture Microplate (Agilent Technologies) coated with Cell‐Tak (Agilent Technologies) and centrifuged for 1 min at 100 g, followed by deceleration without braking. Neutrophils were plated in Seahorse XF Roswell Park Memorial Institute (RPMI) medium containing 1 mM glutamine and 10 mM glucose and incubated in a CO₂‐free environment at 37°C for 1 h. In some cases, glucose‐ and glutamine‐free medium was used to examine the effects of nutrient deprivation on cellular metabolism. The oxygen consumption rate and extracellular acidification rate (ECAR) were measured using the adenosine triphosphate (ATP) Rate kit, Mito Stress Test kit and Glyco Stress Test kits on the XF96 Extracellular Flux Analyser (Seahorse Bioscience) according to the manufacturer's instructions. To determine the effect of CD98 inhibition on mitochondrial metabolism, neutrophils were incubated for 2 h in the presence of 1 μM JPH203 (Tocris) or vehicle control Dimethylsulfoxide (DMSO) before a Mito Stress Test assay was performed.