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. 2020 Nov 2;9:e60151. doi: 10.7554/eLife.60151

Figure 5. p-4E promotes ISR-dependent glutamine addiction.

Isogenic HCT 116 WT and 4E KI cells were subjected to glutamine deprivation (2 to 0 mM), glucose deprivation (4 to 0 mM) or GLS inhibitor (CB-839, 40 µM) treatment, and analyzed at indicated times (0–48 hr). (A) Attached cells were stained with crystal violet at 48 hr. (B) Cell death were analyzed at 48 hr by flow cytometry and (C) quantification of AnnexinV+ cells. Representative flow cytometry plots and results are shown. (D) The indicated proteins were analyzed by western blotting at indicated times after Gln deprivation. (E) Heatmap of indicated transcripts in polysomes at 24 hr were analyzed by RT-PCR, normalized to WT 0 hr (Siegel et al., 2019). (F–G) WT cells were transfected the indicated siRNAs for 24 hr, followed by 24 hr recovery, and subjected to glutamine deprivation. (F) Heatmaps of indicated transcripts in total RNA at 24 hr were analyzed by RT-PCR, normalized to WT 0 hr (Siegel et al., 2019). (G) Cell death at 48 hr was quantitated by Annexin V+ cells. (H) 4EKI cells were transfected with control, Myc or ATF4 expression construct and subjected to glutamine deprivation. Cell death at 48 hr was quantitated by Annexin V+ cells. (I) Cell death 48 hr after GLSi was quantitated by Annexin V+ cells.

Figure 5.

Figure 5—figure supplement 1. p-4E drives Myc and ISR-dependent glutamine addiction.

Figure 5—figure supplement 1.

HCT 116 WT or 4E KI cells were subjected to glutamine deprivation or GLS inhibitor (CB-839, 40 µM) treatment, and analyzed at indicated times (0–48 hr). (A) WT cells were transfected the indicated siRNAs for 24 hr, and followed by 24 hr recovery, then subjected to glutamine deprivation. The indicated proteins at 24 hr were analyzed western blotting. (B) 4EKI cells were transfected with control, Myc or ATF4 (Flag-tagged) expression constructs for 24 hr, followed by 24 hr recovery and subjected to glutamine deprivation. The indicated proteins at 24 hr were analyzed by western blotting. (C) Attached cells were stained with crystal violet at 48 hr, and (D) quantitation of Annex V+ cells by flow cytometry. (E–G) WT and 4EKI2 cells were analyzed for cell growth and death at 48 hr, and the indicated proteins at 24 hr. (I) The indicated protein and (H) transcripts were analyzed 24 hr after GLSi (CB) treatment. Values represent mean+s.d. (n = 3). **p<0.01 (Student’s t-test, two tailed). WT vs. KI.