Fig. 5: Limiting serine availability decreases formate overflow and cancer cell migration.

a MID of intracellular serine in MDA-MB-468 cells upon [U-¹³C]glucose and 24 h 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, 15 μM BI-4916, and 50 nM MTX; mean ± SEM (n = 10 for CTRL; n = 3 for WQ, MTX, WQ MTX, BI, BI MTX; n = 2 for NCT; n = 1 for CBR) of independent experiments each measured in triplicate wells. b Formate M + 1 isotopologue exchange rate in MDA-MB-468 cells using [U-¹³C]glucose and treatment as in (a). Dots indicate independent experiments measured in triplicate wells; mean ± SEM (n = 10 for CTRL; n = 3 for WQ, MTX, WQ MTX, BI, BI MTX; n = 2 for NCT; n = 1 for CBR). c Basal cellular respiration upon 24 h 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, and 15 μM BI-4916 in MDA-MB-468 cells as fold mitochondrial OCR compared to control. Dots represent individual experiments composed of six technical replicates; mean ± SEM (n = 10 for CTRL, n = 7 for WQ, n = 3 for BI, n = 2 for NCT, n = 1 for CBR). d M + 2 isotopologues of selected TCA-metabolites in MDA-MB-468 cells upon [U-¹³C]glucose and treatment as in (b). Dots indicate independent experiments measured in triplicate wells; mean ± SEM (n = 7 for CTRL, n = 3 for WQ, BI, n = 2 for NCT, n = 1 for CBR). e Migration of MDA-MB-468 cells upon 50 nM MTX, 15 μM BI-4916 and f serine- and glycine starvation and respective AUC; mean ± SEM of independent experiments (n = 5 for (e), n = 4 for (f)); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. g mRNA expression from indicated genes in MDA-MB-468 cells upon 72 h 50 nM MTX, 15 μM BI-4916, and serine- and glycine starvation measured by real-time RT-qPCR. Mean ± SEM (n = 4 for BI, BI + MTX, n = 3 for MTX-SG, MTX-SG + BI, n = 2 for -SG, n = 1 for BI-SG). h Cell death induction in MDA-MB-468 cells upon 48 h 50 nM MTX, 15 μM BI-4916 and serine- and glycine starvation was assessed by flow cytometry and AnnexinV-FITC/PI-staining; mean ± SD of independent experiments (n = 3). Two-way ANOVA with Dunnett’s multiple comparisons test. i Migration of MTX-resistant MDA-MB-468 cells upon 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM of independent experiments (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. j Cytosolic and mitochondrial compartments of 1C metabolism and the pharmacologic intervention points of MTX and inhibitors of PHGDH. Source data are provided as a Source Data file.