Hypoxia modulates apoptosis, cell cycle, and metabolism in T-ALL. (A) Growth of T-ALL in conditions of low and high O₂ levels. Shown are the numbers of leukemic cells after 4 days of culture in normoxia and hypoxia expressed as fold variation compared with number of cells plated at day 0 in contact with MS5-DL1 cells. Every dot is the mean of technical triplicates for 1 experiment. T-ALL #1, n = 6exp; T-ALL #2, n = 6exp; T-ALL #3, n = 4exp; T-ALL #4, n = 3exp; and T-ALL #5, n = 2exp. (B) Apoptosis levels for T-ALL in culture at high and low O₂ levels. Shown are fold variation of apoptotic cells relative to control, gated on lymphocyte cells. Every experiment was done in technical triplicate. T-ALL #1, T-ALL #2, T-ALL #3, and T-ALL #4, n = 2exp for each T-ALL. (C) Cell cycle analysis of T-ALL in high and low O₂ levels. (i) Representative plots of Ki67/Hoechst staining of T-ALL #1. Proportion of Ki67⁺ leukemic cells (ii) and proportion of cells in G₀, G₁, S, and G₂/M phases (iii). Shown are mean ± SEM of cultures with T-ALL #1 and T-ALL #3, n = 6exp. (D) Metabolic status of T-ALL in high and low O₂ levels. MTG (i) and TMRE (ii) staining of a representative experiment (T-ALL #2) and MFI from T-ALL cultured in hypoxia reported to controls. Every experiment was done in triplicate. T-ALL #1, T-ALL #2, T-ALL #4, and T-ALL #5, n = 1exp for each T-ALL except for T-ALL #1, which was n = 2exp. (E) Lactate levels in T-ALL medium cultured in hypoxia. The blasts were cultured for 3 days in hypoxia or normoxia. The measure of lactate levels was performed on the culture medium according to the manufacturer protocol (lactate assay kit: 03183700; Roche). Shown are mean ± standard deviation of lactate triplicate values. One experiment was done with each leukemia sample. (F) Relative mRNA expression levels of HIF-1α, Glut3, VEGF, and CXCR4 genes. Tested on T-ALL #1, T-ALL #2, T-ALL #3, and T-ALL #4, n = 9exp. Statistics were done using the Friedman test: *P < .05, **P < .01, ***P < .001. exp, experiment.