Fig. 5: Anti-NUPR1 sdAb#07.81 induces premature senescence and derepresses ferroptosis.

A Representative light microscopy images of GLB1 staining in 4T1 added with purified sdAb-Con, anti-NUPR1 sdAb#07.81 protein or NUPR1 inhibitor ZZW-115 at the indicated concentration for 48 h (n = 3), Scale bar, 200 μm. B Representative light microscopy images of GLB1 staining in MDA-MB-231 (Con) or sgNUPR1, or MDA-MB-231 added with purified sdAb-Con, anti-NUPR1 sdAb#07.81 protein or ZZW-115 at the indicated concentration for 48 h (n = 3), Scale bar, 200 μm. C Representative light microscopy images of GLB1 staining in MDA-MB-231 treated with chloroquine (CQ) or not, and added with purified sdAb-Con or anti-NUPR1 sdAb#07.81 protein for 48 h (n = 3), Scale bar, 200 μm. D MDA-MB-231 cells were subjected to triple IF with anti-SQSTM1 mouse (green), anti-LC3B rabbit (red), and DAPI (blue), followed by visualization with confocal microscopy. White arrows showed the yellow points referring to co-localization. Purified sdAb-Con, sdAb#07.81 proteins or ZZW-115 were added to MDA-MB-231 cells, and IF was performed after 48 h. Scale bars: 100 μm. E, F Lipid peroxidation measurement via C11-BODIPY live-cell confocal imaging in 4T1 and MDA-MB-231 cells following 24 h treatment with anti-NUPR1 sdAb#07.81, RSL3, and sdAb#07.81 or ZZW-115. Three independent experiments were performed. G Establishment of stable LCN2 overexpression and knockdown MDA-MB-231 cell Lines. The levels of Flag and LCN2 were measured using an immunoblot. ACTB used as a loading control. H Cell growth of MDA-MB-231 cells was measured by CCK-8 assay. Data are presented as the mean ± SEM of three independent experiments; the error bars show one standard deviation. ****P < 0.0001.