Fig. 3.

Notch1 upregulation induced glutamine addiction in T‐ALL cells. (A) RNA levels of NICD, as estimated by quantitative PCR, of EV and NICD H33HJ‐JA1 cells cultivated in complete medium. (B) Luciferase‐dependent luminescence was estimated in EV and NICD cells using a luminometer. (C) RNA content of EV and NICD cells was extracted from cells cultivated in complete medium. MYC, HES1, and HEY1 mRNA levels were estimated by quantitative PCR. (D‐E) Growing curves of EV and NICD cells incubated in the presence (D) or absence (E) of glutamine for the indicated times. Cell concentration was determined using a cell counter. (F) Cell death of EV and NICD cells incubated either in the presence (+Q) or absence (−Q) of glutamine during 72 h. Cell death was estimated using a trypan blue assay. (G) Cell viability, as estimated by trypan blue assay, of EV and NICD incubated in the absence of glutamine for the indicated time. (H) Western blot analysis of the apoptotic markers cleaved PARP and cleaved caspase 3 of EV and NICD cells incubated as in F. (I) Late apoptotic cell percentage, as quantified by flow cytometry analysis of PI and annexin V content, of EV and NICD cells incubated as in F. Graphs show mean values ± SEM (n ≥ 3, *P < 0.05). Two‐way ANOVA followed by Bonferroni's comparison as a post hoc test was used to evaluate the statistical difference between more than two groups. t‐Test analysis was used to evaluate the statistical difference between two groups.