Multi-Omic Analysis Reveals Independent Glucose and Glutamine Activation–Braking Systems in Cancer Cells : Supplementary Data

Supplementary Information

Supplementary File 1 | Differential gene expression under combined nutrient deprivationDifferential expression analysis comparing glucose–glutamine–deficient conditions (−Glc/−Gln) with complete medium (+Glc/+Gln) in MDA-MB-231 cells.Tab A: Genes significantly upregulated under nutrient deficiency (FDR < 0.05, log₂FC ≥ 1.5).Tab B: Genes significantly downregulated under nutrient deficiency (FDR < 0.05, log₂FC ≤ −1.5).Average normalized expression values and associated statistical metrics are provided.

Supplementary File 2 | Pathway and transcription factor enrichment under nutrient deficiencyFunctional enrichment analyses of genes differentially expressed under glucose–glutamine deficiency.Tab A: Reactome and Gene Ontology pathway enrichment for genes upregulated under deficiency.Tab B: Transcription factor and pathway enrichment for genes downregulated under deficiency, highlighting suppression of cell-cycle, chromatin licensing, and biosynthetic programs.

Supplementary File 3 | Glucose-specific normalization of deficiency-induced transcriptional programsGenes whose deficiency-induced expression changes are selectively normalized by glucose-only supplementation.Tab A: Genes significantly upregulated under deficiency and subsequently downregulated toward control levels exclusively by glucose-only supplementation (Deficient vs Glucose-only: FDR < 0.05, |log₂FC| ≥ 1.5), but not by glutamine-only supplementation.Tab B: Genes significantly downregulated under deficiency and selectively restored by glucose-only supplementation.Average normalized expression values across all conditions are shown.

Supplementary File 4 | Glutamine-specific restoration of metabolic infrastructure programsGenes selectively restored by glutamine-only supplementation following nutrient deficiency.Tab A: Genes significantly downregulated under deficiency and significantly upregulated upon glutamine-only supplementation (Deficient vs Glutamine-only: FDR < 0.05), but not by glucose-only supplementation.Tab B: Functional enrichment analysis of glutamine-restored genes, highlighting glycolytic capacity, lipid biosynthesis, membrane remodeling, and metabolic infrastructure pathways.

Supplementary File 5 | Nutrient-dependent transcriptional braking systemsGenes defining active, nutrient-specific transcriptional braking programs independent of the canonical nutrient stress response.Tab A (Glutamine braking systems): Genes stable between complete and deficient conditions that are selectively downregulated upon glutamine-only supplementation, indicating glutamine-dependent repression of nuclear organization, DNA replication readiness, chromatin regulation, and RNA processing programs.Tab B (Glucose braking systems): Genes stable between complete and deficient conditions that are selectively downregulated upon glucose-only supplementation, indicating glucose-dependent repression of lipid flux, sterol biosynthesis, mitochondrial redox throughput, and vesicular trafficking programs.Average normalized expression values across all conditions are provided.

Supplementary File 6 | Targeted metabolomic and lipidomic profiling across nutrient conditions

6A | Targeted metabolite changes across defined nutrient conditionsTargeted metabolites quantified from conditioned media at 36 h and annotated by nutrient responsiveness across full, deficient (−Glc/−Gln), glucose-only, and glutamine-only conditions. Metabolites are classified by directional enrichment relative to nutrient deficiency and used for directional validation of discovery metabolomics.

6B | Ranked metabolite abundance defining nutrient-enforced metabolic statesMetabolites rank-ordered from highest to lowest relative abundance within each nutrient condition. This analysis highlights dominant metabolite pools characteristic of each nutrient-enforced metabolic state rather than differential abundance.

6C | Orthogonal validation of extracellular metabolites and pH measurementsIn-house validation assays performed on conditioned media at 36 h corresponding to Figure 6. Extracellular glucose and lactate were quantified using enzymatic colorimetric assays, and extracellular pH was measured using a calibrated pH probe. Values represent the mean of three independent measurements per condition (n = 3) and confirm glucose-dependent lactate production and glutamine-driven, lactate-independent extracellular acidification.