Extended Data Fig. 2: GS depletion inhibits tumor growth without rewiring metabolic pathways.
From: Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth
a, Flow cytometry analysis showing apoptosis of H460 cells transduced with control (shScram) or GS-targeting shRNAs (shGS-1 and shGS-2) cultured in glutamine-deprived media. b, Quantification of intracellular glutamine and glutamate in H460 cells transduced with control shRNA or GS-targeted shRNAs in the presence of 2 mM glutamine. (n=3 independent biological replicates). c, A schematic showing the major metabolites involved in the glutamine metabolism network. d, Quantification of the content of GSH and GSSG, components of the TCA cycles, nucleotides and hexosamines in H460 cells transduced with control shRNA or GS-targeted shRNAs in the presence of 2 mM glutamine measured by mass spectrometry analysis. (n=3 independent samples). e, Expression of GS in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C. f. The catalytic activity of GS in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 0.5 mM glutamine. g, Relative cell survival of H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 0.5 mM glutamine. h, Colonies formed by A549 (left) and Huh7 (right) cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 2 mM glutamine. (n=3 independent biological replicates for f-h). i, In vivo tumor growth of H460 cells (left) and Huh7 cells (right) transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C by weight. (n=16 for each group of the H460 tumors; for Huh7 tumors, n=8 for the shScram and shGS-1+GS-WT tumors, n=7 for shGS-1 and shGS-1+GS-R324C). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for b, d, f, g, h, and i (right panel). Mann-Whitney test was applied for i (left panel). *: p<0.05, **: p<0.01.
