MondoA confers adaptation to metabolic stress, induces glutamine anaplerosis, and limits ROS production in B-ALL cells. (A) Cellular ROS measurement of Nalm6 MKO and control cells under either glutamine (GLN) withdrawal or full media. 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence measured by flow cytometry (n = 3 for each experimental condition, P < .001, 1-way ANOVA with the Bonferroni posttest). (B) Cell death rate determined by Annexin V and 7-aminoactinomycin D (7AAD) staining in control and MKO cells in either full medium or medium lacking GLN (n = 3 for each experimental condition, P < .05, P < .01, or P < .001, 1-way ANOVA with the Bonferroni posttest). (C) PDH activity assessed by NADH production. PDH activity in MKO and MOE Nalm6 cells with or without GLN starvation assessed by colorimetric PDH activity assay kit (n = 3 for each time point, P < .001, 2-way ANOVA with the Bonferroni posttest). (D) Western blot showing MondoA, PARP and cl-PARP, and pPDH and total PDH in control Nalm6 cells and cells with MondoA overexpression under 16 hours of GLN starvation and oligomycin (2.5 µM) or SBI477 (10 µM) treatment. GAPDH as loading control. (E) Relative cell viability after 16 hours of GLN starvation and αKG (5 mM). GLN (4 mM) readdition as a control as measured by Celigo S Imaging Cytometer (Nexcelom Bioscience). Live cells were measured by costaining cells with Hoechst 33342 to identify nuclei and fluorescently labeled CalceinAM (C3099; Thermo Fisher Scientific) to identify live cells. (F) Western blot showing MondoA, PARP, and cl-PARP in control Nalm6 cells, MKO and cells with MondoA overexpression under 16 hours of GLN starvation and asparagine (0.1 mM). GAPDH as loading control. (G) Western blot showing MondoA, PARP, and cl-PARP in control Nalm6 cells, MKO, and cells with MondoA overexpression under 16 hours of GLN starvation and glucose (20 mM). GAPDH as loading control. MFI, mean fluorescence intensity. *P < .05; **P < .01; ***P < .001.