FIGURE 1.

Effects of amino acid (AA) deprivation on Me-AIB uptake, SNAT2 mRNA, and SNAT2 protein abundance in HeLa cells. (A) HeLa cells were incubated in the absence or presence of 1× physiological AA mix for 6 h and then subsequently maintained or re-exposed to a 1× AA mix for times indicated prior to analysis of Me-AIB uptake. (B) Cells were incubated in the absence or presence of 1× physiological AA mix for 8 h or maintained in buffer in which the AA complement was substituted with 2 mM Me-AIB. Cells were subsequently either used for analysis of 10 μM Me-AIB uptake or lysed for analysis of SNAT2 protein abundance by immunoblotting with GAPDH used a loading control. (C) HeLa cells were transiently transfected with a pcDNA3 luciferase reporter construct, driven by the SNAT2 promoter; 48 h post-transfection cells were incubated in the absence (8 h) or presence of AAs. SNAT2 promoter activity was then determined by the analysis of luciferase activity (luminescence quantification). Results calculated as relative luminescence (arbitrary units) per microgram of protein and normalized as a fold change to a control value. Alternatively, following AA deprivation of cells SNAT2 mRNA was quantified using RT-PCR. (D) HeLa cells were AA deprived for 8 h and uptake of 10 μM [14C]-Me-AIB subsequently assessed in absence or presence of indicated AAs at 2 mM. Values are mean ± SEM from three to six separate experiments with each uptake measure being assayed in triplicate in individual experiments. The asterisks indicate a significant difference from indicated bars P ≤ 0.05.