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. 2020 Mar 10;28(5):1339–1358. doi: 10.1016/j.ymthe.2020.03.003

Figure 7.

Figure 7

Glycine Promotes mTORC1 Translocation via v-ATPase and RagB

(A) Western blot to detect phosphorylated S6K1, S6, and 4EBP1 expression in starved C2C12 cells followed by re-stimulation of total amino acids and glycine. 30 μg of total protein was loaded and tubulin was used as a loading control. AA+ refers to the supplementation of total amino acids; Gly+ refers to the supplementation of glycine into starved cells; AA− represents the depletion of total amino acids. (B) Quantitative analysis of the ratio of phosphorylated S6K1, S6, and 4EBP1 to total expression of counterparts (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (C and D) Immunocytochemistry for mTOR (C) and quantitative analysis of cells with mTOR at lysosomes (D) in starved C2C12 cells followed by re-stimulation of total amino acids or glycine (scale bar, 10 μm) (n = 10; one-way ANOVA and post hoc Student-Newman-Keuls test). LAMP1 and LAMP2 were used as lysosome markers. (E and F) Immunocytochemistry for mTOR (E) and quantitative analysis of cells with mTOR at lysosomes (F) in HEK293T cells treated with glycine and BAF (scale bar, 10 μm) (n = 10; two-tailed t test). (G) Western blot to detect phosphorylated S6K1 and S6 expression in HEK293T cells treated with glycine or glycine and BAF. 30 μg of total protein was loaded and tubulin was used as a loading control. (H) Western blot to detect phosphorylated S6K1 and S6 expression in RagB knockout HEK293T cells treated with glycine or total amino acids. 30 μg of total protein was loaded and GAPDH was used as a loading control. RagB CON refers to normal HEK293T cells; RagB KO means RagB knockout HEK293T cells. Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.