Figure 7. Glu-derived Ala stimulates mTORC1 and promotes Hep3B cell proliferation.

(A) Hep3B cells prestarved in MEM or EBSS for 8 hours were left unstimulated (Ctrl) or stimulated for 30 minutes with 1× or 2× NEAA mix. Immunoblotting was performed. (B) Cells stably expressing sgCtrl and sgGLUL were prestarved in MEM followed by 30-minute treatment with 1× NEAA mix. Immunoblotting was performed. (C) sgCtrl and sgGLUL cells in various amounts of Gln supplied with 0 or 1× NEAA mix. Relative cell growth is shown. (D) Cells prestarved in MEM for 4 hours were left unstimulated (Ctrl) or stimulated for 30 minutes with 1 mM indicated amino acids or a 1× NEAA mix. Cells cultured in full medium (MEM + 10% FBS + 1× NEAA mix) were used as a positive control. (E) Cells were cultured in 0 or 0.2 mM Gln supplied with individual NEAAs or a 1× NEAA mix. Relative growth compared with the control is shown. (F) sgCtrl and sgGLUL cells were transfected with indicated individual siRNAs or a mix of all 4 siRNAs (siMIX4) for 48 hours. Cells were cultured in MEM for 9 hours and harvested. (G and H) Indicated cells were prestarved in MEM for 8 hours, and then were treated with NH₄Cl for 24 hours (G) or with 1.5 mM dmKG plus 4 mM NH₄Cl for 1 hour (H). (I) Cells transfected with siControl, siGPT1, or siMIX4 for 48 hours were cultured in MEM containing 0.2 mM Gln without or with dmKG plus NH₄Cl for 72 hours. Relative cell growth is shown (n = 3). (J) sgCtrl and sgGLUL cells were cultured in MEM containing 0 or 0.2 mM Gln and supplied with dmKG plus NH₄Cl. Relative growth is shown (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-tailed t test (C and J) or 1-way ANOVA (E and I).