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. 2021 Jan 14;12:366. doi: 10.1038/s41467-020-20223-y

Fig. 6. Combining PHGDH inhibition with serine/glycine depletion inhibits global protein synthesis.

Fig. 6

a Cells were grown in CM or -SG medium supplemented or not with 10 µM PH755 for 24 h. Western blots show Phospho-GCN2 (Thr899), GCN2, Phospho-eIF2α (Ser51) and eIF2α. Membranes were reprobed with vinculin as a loading control. Data are representative of two independent experiments. b Cells were grown in CM or -SG medium supplemented or not with 10 µM PH755 for 6 h or 24 h. Relative gene expression of ATF4 and PHGDH were measured by qPCR and normalised to the cells grown in CM for 6 h. Data are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA with Tukey’s post hoc test). c Cells were grown in CM or -SG medium supplemented or not with 10 µM PH755 for 24 h. Puromycin (90 µM) was added in culture medium 10 min before harvesting the cells. When indicated, cells were treated with 10 µg/mL cycloheximide (CHX), a well-known protein synthesis inhibitor, 5 h before harvesting the cells. Western blots show puromycylated peptides. Membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments. d Cells were grown in CM or -SG medium supplemented or not with 10 µM PH755 for 24 h. When indicated, cells were treated with 10 µM MG-132, a proteasome inhibitor, 6 h before harvesting the cells. Western blots show the expression of c-MYC, HIF1α and p53. Membrane was reprobed with vinculin as a loading control. Data are representative of three independent experiments. e Cells were grown in CM, -SG medium or -SG medium +10 µM PH755 supplemented or not with 1 mM sodium formate plus 0.4 mM glycine (For/Gly). Western blot shows the expression of the three SSP enzymes PHGDH, PSAT and PSPH or the expression of ATF-4 and its canonical target ASNS after a 24 h incubation in these medium. Membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments.