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. 2020 Jul 13;36:101643. doi: 10.1016/j.redox.2020.101643

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© 2020 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 2 — Glutamine is indispensable for cancer cell survival under ECM detachment. (A) ATP levels in HepG2, HeLa, and HT1080 cells cultured in complete medium, glucose-deficient (Glc (-)), glutamine-deficient (Gln (-)), or glucose and glutamine-deficient (Neither) medium for 24 h under attached (AT) or detached (DT) conditions. (B) Full-length PARP (F-PARP) and cleaved PARP (C-PARP) expression in HepG2 cells cultured as indicated in panel A for 24 h. β-actin served as a loading control. (C) Viability of HepG2 cells cultured for 24 h in the indicated medium on adherent or poly-HEMA-coated plates. (D) ATP levels in HepG2 cells cultured in the indicated nutrient-deficient or supplemented medium on attachment or detachment plates for 24 h. Complete: DMEM-based complete nutrient medium; AA: DMEM-amino acid; Vit: DMEM-vitamin; All deficient: deficient for all nutrients. (E) F-PARP and C-PARP expression in HepG2 cells cultured in the indicated nutrient-deficient or supplemented medium on attachment or detachment plates for 24 h. (F) F-PARP or C-PARP expression in HepG2 cells cultured in complete medium or in all nutrients-free medium supplemented with 2 mM glutamine and the indicated doses of cystine for 24 h under detached conditions. α-tubulin served as a loading control. (G) ROS (H₂O₂) and (H) ATP levels in HepG2 cells cultured in complete medium or all nutrients-free medium supplemented with 2 mM glutamine and the indicated dose of cystine for 24 h in detached conditions. (I) H2AX phosphorylation (p-H2AX) and PARP expression in whole-cell extracts of HepG2 cells incubated with or without 1 mM BSO for 24 h under attached or detached conditions. β-actin served as a loading control. (J) ATP levels, (K) ROS levels, (L) H2AX phosphorylation and PARP expression in HepG2 cells cultured in glutamine-free medium in the presence (Cont) or absence (-) of glutamine, with or without 4 mM DM-αKG under detached conditions. β-actin served as a loading control. Representative western blots are shown. Data are presented as mean ± SD of three independent sets of experiments. NS, not significant; **P < 0.01; ***P < 0.001.

Fig. 2 — Glutamine is indispensable for cancer cell survival under ECM detachment. (A) ATP levels in HepG2, HeLa, and HT1080 cells cultured in complete medium, glucose-deficient (Glc (-)), glutamine-deficient (Gln (-)), or glucose and glutamine-deficient (Neither) medium for 24 h under attached (AT) or detached (DT) conditions. (B) Full-length PARP (F-PARP) and cleaved PARP (C-PARP) expression in HepG2 cells cultured as indicated in panel A for 24 h. β-actin served as a loading control. (C) Viability of HepG2 cells cultured for 24 h in the indicated medium on adherent or poly-HEMA-coated plates. (D) ATP levels in HepG2 cells cultured in the indicated nutrient-deficient or supplemented medium on attachment or detachment plates for 24 h. Complete: DMEM-based complete nutrient medium; AA: DMEM-amino acid; Vit: DMEM-vitamin; All deficient: deficient for all nutrients. (E) F-PARP and C-PARP expression in HepG2 cells cultured in the indicated nutrient-deficient or supplemented medium on attachment or detachment plates for 24 h. (F) F-PARP or C-PARP expression in HepG2 cells cultured in complete medium or in all nutrients-free medium supplemented with 2 mM glutamine and the indicated doses of cystine for 24 h under detached conditions. α-tubulin served as a loading control. (G) ROS (H₂O₂) and (H) ATP levels in HepG2 cells cultured in complete medium or all nutrients-free medium supplemented with 2 mM glutamine and the indicated dose of cystine for 24 h in detached conditions. (I) H2AX phosphorylation (p-H2AX) and PARP expression in whole-cell extracts of HepG2 cells incubated with or without 1 mM BSO for 24 h under attached or detached conditions. β-actin served as a loading control. (J) ATP levels, (K) ROS levels, (L) H2AX phosphorylation and PARP expression in HepG2 cells cultured in glutamine-free medium in the presence (Cont) or absence (-) of glutamine, with or without 4 mM DM-αKG under detached conditions. β-actin served as a loading control. Representative western blots are shown. Data are presented as mean ± SD of three independent sets of experiments. NS, not significant; **P < 0.01; ***P < 0.001.