Figure 7. Cell proliferation inhibition by combining sub-effective doses of ACSL4 and mTOR pathway inhibitors.

MCF-7 Tet-Off empty vector and MCF-7 Tet-Off/ACSL4 cells were plated at a density of 4000 cells/well in 96-well plates with 10% FBS-supplemented D-MEM medium and allowed to adhere overnight at 37°C in a humidified 5% CO₂ atmosphere. The medium was then changed to serum-free medium. After 24 h, the cells were switched to 10% FBS-supplemented D-MEM medium with rapamycin (10 nM) and/or rosiglitazone (75 μM) for 96 h. Subsequently, cell proliferation was measured by BrdU incorporation assays as described before [6]. Data are presented as inhibition of cell proliferation compared to control cells. White bars indicate a single inhibitor treatment while grey bars indicate combined inhibitor treatment. Data are presented as means ± SD. a: ***p < 0.001 vs. single inhibitors. Inset: MCF-7 Tet-Off/ACSL4 cells were incubated in the presence or absence of rosiglitazone (75 μM) alone or in combination with rapamycin (10 nM) for 48 h. p-S6 protein levels were evaluated by Western blot and a representative blot is shown.