Figure 1. Deletion of the Rb family Enhances Glutamine Uptake and Conversion to Glutamate Via Elevated ASCT2 Expression and GLS1 Activity.

a. Glutamine uptake in WT, Rb-1^−/− and TKO cells was assayed by ¹⁴C-glutamine labeling. Data are represented as nmol / min / mg protein. Mean ± s.d. from triplicate measurements from three independent cell preparations. * p < 0.005. b. Glutamine uptake was determined in Rb-1^−/− and TKO cells following reconstitution of Rb-1 expression. Data are represented as nmol / min / mg protein. Mean ± s.d. from triplicate measurements from three independent cell preparations. * p < 0.01. c. mRNA expression of ASCT2 and SLC38A5 between WT and TKO MEFs. For the ASCT2 and SLC38A5 comparisons, data are represented as relative mRNA with WT levels set to 1. Protein expression of ASCT2 between WT and TKO MEFs is represented as relative protein expression with WT levels set to 1. For protein expression, shown is a representative image from three separate experiments. * p < 0.01. d. For GLS, mRNA levels are relative to expression of WT GLS1 set to 1. For mRNA, shown is mean ± s.d. from three independent RNA preparations. Protein expression of GLS1 between WT and TKO MEFs is represented as relative protein expression with WT levels set to 1. For protein expression, shown is a representative image from three separate experiments. * p < 0.01. e. Secreted ammonia levels were determined in the medium from WT and TKO cells. Data are represented as nmol / min / mg protein, and shown are mean ± s.d. from triplicate measurements from two independent experiments. * p < 0.005. f. Incorporation of glutamine carbon into glutamate was determined by stable isotope labeling of WT and TKO cells with [¹³C₅,¹⁵N₂]-glutamine, as described in the methods. Shown are representative 2-D NMR spectra of peaks representative of glutamate. The boxes connect the ¹³C satellite peaks of glutamate and the central peak denoted ¹²C is from glutamate that contains no ¹³C.