Fig. 2

Effects of OST-01 on LRP8-regulated ferroptosis in TNBC cells. A-B Effects of OST-01 on the expression of key regulators of BCSC. MDA-MB-231 cells were treated with 1 µL/mL of ethanol control or OST-01 (1 µL of OST-01 extract contains 250 µg of dry plant extract) for 24 h. A Left, mRNA levels were measured by qPCR. Right, protein levels were measured by immunoblotting. Quantification of protein expressions are shown on top. B The treated cells were stained with the indicated antibodies; representative confocal images are shown. Scale bar, 10 μm. C Effects of OST-01 on ferroptosis in TNBC cells. The indicated TNBC cell lines (4T1, MDA-MB-231, and BT549) and the triple-positive BC cell line BT474 were treated with OST-01 in a dose-dependent manner for 24 h. Left: Lipid peroxide levels were measured using a lipid peroxidation assay. Middle: Malondialdehyde (MDA) levels were assessed. Right: Glutathione peroxidase (GPX) levels were measured by a GPX activity assay. D Effects of OST-01 on selenoprotein expression in TNBC cells. MDA-MB-231 TNBC cells were treated with OST-01 in a dose-dependent manner for 24 h. Cell lysates were immunoblotted with the indicated selenoprotein antibodies. Quantification of protein expressions are shown on top. E-G Effects of LRP8 overexpression on OST-01-induced ferroptosis and TNBC cell growth inhibition. E MDA-MB-231 TNBC cells with empty vector control or LRP8 overexpression were treated with OST-01 in a dose-dependent manner for 24 h. Left, cell proliferation was assessed. Right, immunoblotting with the indicated antibodies was performed. Quantification of protein expressions are shown on top. F MDA-MB-231 TNBC cells with empty vector control or LRP8 overexpression were treated with OST-01 in a dose-dependent manner for 24 h. Levels of ferroptosis were measured. Left, lipid peroxides levels. Middle, MDA levels. Right, GPX levels. G 10⁶ MDA-MB-231 TNBC cells with empty vector control or LRP8 overexpression were subcutaneously injected into nude mice. After 2 weeks, the transplanted mice were treated with OST-01 (1 µL/g, oral gavage, BID) for 5 weeks. Left, image of tumors. Right, tumor weight (each group, n = 4). H Effects of OST-01 on CD44 and CD24 expression. MDA-MB-231 TNBC cells were treated with 1 µL/mL of ethanol control or OST-01 for 24 h. Left, the treated cells were stained with anti-CD44 (pink) and anti-CD24 (green) antibodies. Images were captured using confocal microscopy. Scale bar, 10 μm. Right, graph showing the changes in CD44 + CD24- and CD44 + CD24 + populations upon OST-01 treatment. I-J Effects of OST-01 on BCSCs in vitro and in vivo. I MDA-MB-231 TNBC cells were treated with OST-01 in a dose-dependent manner for 24 h. Cell lysates were immunoblotted with the indicated antibodies. Quantification of protein expressions are shown on top. J MDA-MB-231 xenograft tumors isolated from mice treated with ethanol control or OST-01 (as described in Fig. 1E) were subjected to frozen sectioning and stained with the indicated antibodies. Images were captured using confocal microscopy. Scale bar, 1 μm. K Schematic model of the mechanism of action of OST-01 on TNBC cells. OST-01 inhibits LRP8 expression, leading to reduced levels of selenoproteins, including GPX4. The decrease in selenoproteins induces ferroptosis, which in turn suppresses the stemness activities of BCSCs. This effect is achieved through the induction of mesenchymal-to-epithelial transition, resulting in enhanced differentiation of the cells