Fig. 3: MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis.

a Interdependence of glycolysis and tricarboxylic acid (TCA) cycle and metabolic ¹³C label pattern from [U-¹³C]glutamine. b Absolute consumption and release (CORE) rates of glutamine (Gln) and glutamate (Glut) in the culture medium of MDA-MB-468 cells after 24 h 50 nM MTX. Dots represent the mean of an individual experiments composed of triplicate wells; mean ± SEM (n = 3). c Enrichment of selected isotopologues of Gln, Glut, α-ketoglutarate (αKG), fumarate (Fum), malate (Mal), aspartate (Asp), and citrate (Cit) upon [U-¹³C]glutamine tracer in response to 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an independent experiments in triplicate wells; mean ± SEM; unpaired, two-tailed t-test with Welch’s correction (n = 3). d Interdependence of glycolysis and TCA cycle and metabolic ¹³C label pattern from [U-¹³C]glucose. e Absolute CORE rates of lactate and glucose in the culture medium of MDA-MB-468 cells after 24 h 50 nM MTX. Dots represent the mean of an individual experiments composed of triplicate wells; mean ± SEM (n = 3). f Enrichment of selcted isotopologues of pyruvate (Pyr), Cit, αKG, Fum, Mal, and Asp upon [U-¹³C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an individual experiments composed of triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction (n = 3). g Basal cellular respiration upon 24 h 50 nM MTX in MDA-MB-468, LN229, and 4T1 cells as quantification of mitochondrial oxygen consumption rate (OCR). Dots represent the mean of an individual experiments composed of six technical replicates; mean ± SEM (n = 3 (LN229), 4 (MDA-MB-468, 4T1)). h Enrichment of M + 3 isotopologue of serine upon [U-¹³C]glucose upon 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an independent experiments in triplicate wells; mean ± SEM; unpaired, two-tailed t-test with Welch’s correction (n = 3). i Ratio of absolute intracellular NAD⁺ to NADH in MDA-MB-468 cells upon 24 h 50 nM MTX. Dots represents the mean of an individual experiments composed of triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction (n = 3). j M + 1 isotopologue of extracellular formate in MDA-MB-468 cells from [U-¹³C]glucose upon 24 h 50 nM MTX. Dots indicate the mean of an independent experiments measured in triplicate wells; mean ± SEM (n = 4). Source data are provided as a Source Data file.