Fig. 2. The Plasmax component sodium selenite prevents ferroptosis and thereby enhances the colony formation capacity of TNBC cells.

(A) Images of colony formation assays performed with MDA-MB-468 cells seeded 500 cells per well and incubated for 14 days in 1:1 DMEM-F12/Plasmax mix or in DMEM-F12 supplemented with the different mixed stock solutions or individual components of Plasmax. Images are representative of three independent experiments. (B) Representative images and (C) quantification of colony formation assays performed with MDA-MB-468 cells seeded at 500 cells per well in DMEM-F12 with different concentrations of Na₂SeO₃ and incubated for 14 days. Means ± SEM; n = 3 independent experiments. (D) Representative images and (E) quantification of the colony formation assays performed with MDA-MB-468 cells seeded at the indicated cell density and incubated for 7 days. Means ± SEM; n = 3 independent experiments. (F) Quantification of colony formation assays performed with MDA-MB-468 cells seeded at 5000 cells per well and incubated for 7 days in DMEM-F12 supplemented with FBS and 50 nM Na₂SeO₃, as indicated. Means ± SEM; n = 3 independent experiments. (G) Representative images and (H and I) quantification of colony formation assays performed with MDA-MB-468 cells seeded at 5000 cells per well and incubated for 7 days in DMEM-F12 with 50 nM Na₂SeO₃, 1 mM N-acetylcysteine (NAC), or 100 μM Trolox, as indicated. Means ± SEM; n = 3 independent experiments. (J) Western blot showing GPX4 levels in MDA-MB-468 cells seeded at low (2000 cells/cm²) or high (10,000 cells/cm²) density and incubated in DMEM-F12 for 3 days with 50 nM Na₂SeO₃, as indicated. Images are representative of three independent experiments. (K) GPX activity measured in MDA-MB-468 cells seeded and incubated as for (J). Means ± SEM; n = 3 independent experiments. (L) Representative images and (M) quantification of colony formation assays performed with MDA-MB-468 cells seeded at 5000 cells per well (L) or at 50,000 cells per well (H) and incubated for 7 days in DMEM-F12 supplemented with 50 nM Na₂SeO₃ and 250 nM (1S,3R)-RSL3. Means ± SEM; n = 2 independent experiments. (N) Lipid peroxidation levels of MDA-MB-468 cells seeded as in (J) and incubated in DMEM-F12 for 48 hours with 50 nM Na₂SeO₃ and for the last hour with 50 μM t-butOOH, as indicated. At the end of the incubation, 1 μM BODIPY 581/591 C11 was added to the cells for 15 min. Means ± SEM; n = 3 or 4 independent experiments. (O) Representative images of MDA-MB-468 cells seeded at 10,000 cells/cm² and incubated in DMEM-F12 with Na₂SeO₃ for 72 hours and for the last 24 hours with 50 μM t-butOOH, as indicated. Images are representative of three independent experiments. (P and R) Representative images and (Q and S) quantification of colony formation assays performed with MDA-MB-468 cells seeded at 5000 cells per well and incubated for 7 days in DMEM-F12 supplemented with 50 nM Na₂SeO₃, 2.5 μM deferoxamine, and 50 nM liproxstatin-1 (Lip-1), as indicated. Means ± SEM; n = 2 (P and Q) or n = 3 (R and S) independent experiments. (T) Schematic representation of the factors affecting the colony-forming capacity of TNBC cells. “X” refers to a factor present in the medium of confluent MDA-MB-468 cells and not yet identified. (F, H, I, K, M, N, Q, and S) Each dot represents an independent experiment, and P values refer to a two-tailed t test for paired homoscedastic samples.