Dexamethasone treatment antagonizes mTORC1 signaling in B-ALL cells. (A) Immunoblot analysis of NALM-6 and MUTZ-5 cells for signaling molecules functioning in the PI3K-AKT-mTORC1 pathway. Cells were treated with 100 nM dexamethasone for indicated hours and then harvested for western blotting with indicated antibodies. (B) NPRL2-WT or NPRL2-KO NALM-6 cells were treated for 24 hours with either DMSO or 100 nM dexamethasone, and activation status of signaling factors functioning in mTORC1 pathways was determined via western blot. (C) Phospho-S6K levels were evaluated by FACS in NPRL2-WT and NPRL2-KO NALM-6 cells after 24 hours of either DMSO or dexamethasone (100 nM) treatment. (D) Either WT RagB or its constitutive-active mutant (RagB Q99L) was overexpressed in NALM-6 cells, and phospho-S6K levels were examined by western blot. (E) Analysis of protein synthesis based on OP-Puro incorporation assays. NPRL2-WT and NPRL2-KO NALM-6 cells were treated 24 hours with either DMSO, cycloheximide (CHX), or 100 nM dexamethasone, and then OP-Puro incorporation was assessed by FACS. CHX, a translation inhibitor, served as a control. Each condition was assessed in triplicate, and data are represented as means ± SD. P values were calculated using ANOVA with Dunnett multiple-comparison test. (F) MUTZ-5 cells were treated with either DMSO or 100 nM dexamethasone for 24 hours with or without TSLP (10 ng/mL), and phospho-S6K levels were assessed by western blot. (G) MUTZ-5 cells were treated for 96 hours with either dexamethasone at various doses (0.32 nM, 1 nM, 3.2 nM, 10 nM, or 32 nM) or DMSO, with or without TSLP (10 ng/mL). Cell viability was assessed as described in Figure 2D. The data presented are representative of multiple independent experiments.