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. 2021 Feb 13;15(5):1412–1431. doi: 10.1002/1878-0261.12877

Fig. 6.

Fig. 6

The lack of GS accumulation induced by Notch1 is responsible for glutamine addiction in Notch1‐positive T‐ALL cells. (A) Cell death, as estimated using a trypan blue assay, of H33HJ‐JA1 cells incubated either in the presence or the absence of glutamine (Q) and MSO (1 mm) during 72 h as indicated. (B) Western blot analysis of the apoptotic markers cleaved PARP, cleaved caspase 3, cleaved caspase 8, and actin of H33HJ‐JA1 cells incubated as in A. (C) Growing curves of H33HJ‐JA1 cells infected with either a plasmid expressing a control nontargeting shRNA (shRNA Control), or a plasmid expressing a shRNA against GS (shRNA GS), and incubated in the absence of glutamine for the indicated times. (D) Western blot analysis of cleaved PARP, cleaved caspase 3, GS and actin of H33HJ‐JA1 cells infected with either a plasmid expressing a control nontargeting shRNA (shRNA Control), or a plasmid expressing a shRNA against GS (shRNA GS), and incubated in the presence or absence of glutamine during 72 h as indicated. (E) Western blot analysis of cleaved PARP, GS and actin of CUTLL1 cells infected with either an empty vector plasmid (pJS27) or with a plasmid overexpressing GS (pJS27‐GS) and incubated either in the presence (+Q) or absence (−Q) of glutamine for 72 h. (F) Western blot analysis of NICD, GS and actin of CUTLL1 and H33HJ‐JA1 cells incubated either in the presence (+Q) or absence (−Q) of glutamine during 72 h as indicated. (G) CUTLL1, HBP‐ALL, MOLT4, H33HJ‐JA1, and LOUCY cells were incubated either in the presence or the absence of glutamine (Q) for 72 h. Cell extracts were collected and levels NICD, GS, and actin were estimated by western blot. (H) CUTLL1 and H33HJ‐JA1 cells were incubated either in the presence (+Q) or absence (−Q) of glutamine. RNA content was extracted and GS mRNA level was estimated by quantitative PCR. (I) H33HJ‐JA1 cells were incubated either in the presence or absence of glutamine (Q) and MG132 (5 µm) during 4 h as indicated. Cell extracts were collected and levels of GS and actin were estimated by western blot. (J) Western blot analysis of GS and actin of EV and NICD cells incubated either in the presence (+Q) or absence (−Q) of glutamine during 72 h as indicated. 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.