Figure 1.

DRAM1 Stimulates Amino-Acid-Induced mTOR Activation

(A) Saos2 TetOn-DRAM1-myc-his-tagged cells were, where indicated, treated with doxycycline (Dox) for 24 h and then starved for 3 h in EBSS before treatment with EBSS containing essential amino acids for the indicated times. mTOR activation was evaluated by measuring phospho-S6 kinase levels by western blot. Total S6 kinase levels or actin were used as loading controls. DRAM1 expression was detected using an anti-Myc-tag antibody.

(B) Dram1^flox/flox MEF expressing Cre recombinase (−/−) or a control vector (fl/fl) was starved for 3 h in EBSS prior to 20 min in EBSS containing 0.8 mM leucine. mTOR activation was evaluated by measuring phospho-S6 kinase, phospho-4E-BP1 levels by western blot. S6K, 4E-BP1, and ERK2 were used as loading controls.

(C) Flow cytometry analysis of cell size of Dram1^flox/flox MEF expressing Cre recombinase (−/−) or a control vector (fl/fl) grown under control or starvation conditions for 3 h. FSC, forward scatter. Boxplot and whiskers: 1–99 percentile. Bar represents median. ^∗p < 0.05.

(D) Cell proliferation of Dram1^flox/flox MEF expressing Cre recombinase (−/−) or a control vector (fl/fl). Equal cell numbers were split on day 0 in complete DMEM. From day 2, cells were harvested daily and counted using Innovatis cell counter. Result shown is representative of 3 independent experiments. Data are mean ± SD. ^∗p < 0.05.

(E) Dram1^flox/flox MEF expressing Cre recombinase enzyme (−/−) or a control vector (fl/fl) were starved for 3 h in EBSS containing glutamine prior to DMEM treatment for the indicated times. Repression of autophagy by mTOR activation was assessed by western blot of LC3B (I and II) and phospho-S6 kinase. ERK2 was used as a loading control. Result shown is representative of 3 independent experiments.

See also Figures S1 and S2.