Dulbecco modified eagle medium (dmem)

The human breast cancer cell lines MCF-7, MDA-MB-231, and Hs 578T were cultured in DMEM (United States Biological) with 10% fetal bovine serum (FBS, Life Technologies), 1% penicillin/streptomycin (P/S), and 1% non-essential amino acids at 37°C and 5% CO₂ in an incubator. The human breast cancer cell line HCC 1937 was cultured in RPMI 1640 (United States Biological) with 10% FBS and 1% P/S. To prepare the culture medium for each amino acid starvation condition, DMEM or RPMI 1640 medium without amino acids (D9800-13 and R9010-01, United States Biological) was mixed with different amino acid combinations lacking a specific amino acid (LAA21-1KT, Sigma-Aldrich).

All reagents not specified were purchased from Sigma (St. Louis, MO, USA). CBR-5884, a PHGDH inhibitor, and RA 839, a Nrf2 (nuclear factor erythroid 2-related factor) activator, were purchased from Tocris (Bristol, UK). LNT-229, LN-308, LN-428 and G55 cells have been described.^{35 (link)} LNT-229 and LN-308 cells were a kind gift from N. de Tribolet (Lausanne, Switzerland), G55 cells were a kind gift from Manfred Westphal and Kathrin Lamszus (Hamburg), LN-428 cells and LN-464 cells were a kind gift from Monika Hegi (Lausanne). MDA-MB-231 and MDA-MB-464 cells were a kind gift from Winfried Wels (Frankfurt, Germany).
Wildtype cell lines were maintained as described.^{35 (link)} For experiments glycine- and serine-free DMEM (US Biological Life Sciences, catalog no. D9800-03/D9802-01, Salem, MA, USA) was supplemented with glucose or serine as indicated. FCS included serine and its supplementation yielded serine concentrations of 20 µM (in comparison to 400 µM when serine was replenished). pLKO.1- and pTetOne-transfected cells were maintained in medium with 2 µg/ml puromycin. 0.1 µg/ml doxycycline was added to induce gene expression of PHGDH from pTetOne-transfected cells. To compare sub cell lines equal cell densities were confirmed by crystal violet (CV) staining as described.^{36 (link)}