Figure 9

Amino acid and glucose deficiencies up-regulate the activity of -575 p27 (p27-5'UTR). The MDA-MB-231 cells were transfected with -575 p27 (p27-5'UTR) and then maintained in 0.2% FBS DMEM for 24 hours. At this point in time, the medium was changed to a DMEM Labeling Kit purchased from Chemicon International (Temecula, CA, USA). This was a deficient basal DMEM supplemented with appropriate components to prepare complete DMEM and DMEM without D-(+)-glucose, L-leucine, L-methionine, L-cysteine, or combination of L-methionine and L-cysteine. Complete DMEM contained 4.5 g/L D-(+)-glucose, 105 mg/L L-leucine, 30 mg/L L-methionine, and 48 mg/L L-cysteine. The cells were maintained in these complete and deficient DMEMs for 24 hours. All assays were performed in triplicates and the transfection experiments were repeated three times.