Extended Data Fig. 1: Glutamine activates SREBP-1 to promote cell proliferation.
a, Heat map comparison of metabolic and overall pathways based on RNA-seq data from H1299 cells under glucose, glutamine or a combination of glucose and glutamine vs. both free conditions (12 h) using the bioinformatics Ingenuity Pathway Analysis (IPA). #NUM, no activity pattern available. b, c, Western blot analysis of cell lysates of cells stimulated with glutamine for 12 h (b) or with 4 mM glutamine at the indicated times (c) under serum-free conditions (glucose 5 mM). d, Lipids derived from 14C-labeled glucose (0.5 μCi, 2 h) in cells after culturing cells with/without glutamine (4 mM) for 12 h in serum-free medium containing 5 mM non-labeled glucose. The results are presented as mean ± SEM (n = 3). e, Proliferation of cancer cells cultured in medium supplemented with 1% dialyzed FBS with/without glutamine (4 mM) or glucose (5 mM) (mean ± SD, n = 3). f, g, Western blot analysis of cells after infection with shRNA-expressing lentivirus for 48 h and then placed in fresh medium (5 mM glucose) with/without glutamine (4 mM) for another 12 h (left panels). Cell proliferation was determined under 1% dialyzed FBS (right panels). The results are shown as mean ± SD (n = 3). h, Western blot analysis of cells after treatment with atorvastatin (5 μM) for 12 h in 5% lipoprotein-deficient serum (LPDS) containing 5 mM glucose with/without glutamine (4 mM). i, Western blot analysis of cells after stimulation with EGF (20 ng/ml) for 12 h in serum-free medium (5 mM glucose) with/without glutamine (4 mM). j, Western blot analysis of cells after incubation with/without aspartate (0.15 mM), asparagine (0.38 mM), leucine (0.38 mM), methionine (0.1 mM), threonine (0.17 mM) or glutamine (2 mM) for 12 h in HBSS buffer (containing 5.6 mM glucose) supplemented with essential amino acids. The dose selected for each amino acid is same as their concentration included in RPMI 1640 medium. Significance was determined by unpaired and two-tailed Student’s t-test (d) or two-way ANOVA with Dunnett’s (e) or Tukey’s (g) multiple comparisons adjustment.
