Stimulation of mTORC1 with L-leucine Rescues Defects Associated with Roberts Syndrome
Figure 1
Human RBS cells showed severely slow proliferation and decreased protein translation.
(A). Immortalized WT, RBS, and corrected cells were seeded into 6-well plates with 0.5×105 cells/mL. After 8 days in culture, immortalized RBS cells showed very poor proliferation, compared with wild type (WT) and ESCO2-corrected (Cor) cells (P<0.01). Inhibition of p53, by Pifα (10 µM) incubation, partially rescued proliferation of RBS cells (P<0.05, 2-way ANOVA). (B). By FACScan analysis, RBS cells accumulated in G2/M phase. (C). Untransformed RBS and normal human skin fibroblasts (HSF) and amniotic fluid-derived cells (AFC) were grown in DMEM plus 15% FBS. 3H-uridine (5 µCi) was incubated with 106 cells from each group for two hours. Total RNA was isolated with TriZol reagent (Invitrogen, U.S.A) and the concentration of each RNA sample was measured by OD260/280. 1 µg of each sample was counted in a Beckman LS 6500 multipurpose scintillation counter to determine the amount of 3H-uridine incorporated. Four independent cultures were labeled to derive the standard deviation. Significance relative to normal cells was calculated using an unpaired t test. P = 0.0039, HSF ESCO2-Mut vs HSF ESCO2-WT; P = 0.000017, AFC ESCO2-Mut vs AFC ESCO2-WT. (D). Equal numbers of untransformed RBS and normal HSF and AFC cells were grown in DMEM plus 15% FBS. Cells were washed in PBS twice, switched to 3 mL Met/Cys-free Dulbecco's modied Eagle's medium containing 10 µM MG-132, a proteasome inhibitor, and pulsed with 30 µCi of 35S-methionine for 4 hrs. Cells were lysed in RIPA buffer and proteins were precipitated by the addition of hot 10% TCA. After centrifugation, the precipitate was washed twice in acetone. The precipitate was dissolved in 100 µL of 1% SDS and heated at 95°C for 10 min. An aliquot of the SDS extract was counted in Esoscint for 35S radioactivity in a liquid scintillation spectrometer to determine the amount of 35S-methionine incorporated into proteins. P = 0.00086, HSF ESCO2-Mut vs HSF ESCO2-WT; P = 0.0005, AFC ESCO2-Mut vs AFC ESCO2-WT.