Fig. 5: USP24 inhibits ferroptosis by increasing the protein stabilization of DHODH. | Cell Death & Disease

Fig. 5: USP24 inhibits ferroptosis by increasing the protein stabilization of DHODH.

From: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

Fig. 5: USP24 inhibits ferroptosis by increasing the protein stabilization of DHODH.The alternative text for this image may have been generated using AI.

A, B Endogenous USP24 and DHODH interactions were detected by coimmunoprecipitation using USP24 and DHODH antibodies, respectively, in MDA-MB-231 and MDA-MB-468 cells. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. C, D The intracellular localization of USP24 (red) and DHODH (green) in MDA-MB-231 and MDA-MB-468 cells was examined by immunofluorescence staining using USP24 and DHODH antibodies and visualized by fluorescence microscopy. DAPI was used to stain nuclei. Scale bars: 5 μm. Immunofluorescence colocalization was quantified using the Manders’ coefficient. E, F The indicated MDA-MB-231 cell lines were pretreated with MG132 (10 μM) for 24 h, and then the extracts were immunoprecipitated with anti-DHODH antibodies and immunoblotted with anti-ubiquitin (Ub), anti-K48-linked ubiquitin, and anti-DHODH antibodies. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, ****P < 0.0001. ns no significance. G, H MDA-MB-231 and MDA-MB-468 cells stably transfected with USP24 shRNA#1 or control shRNA were stably expressed with Flag-DHODH or control vector. USP24 and DHODH expression in cells was measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns no significance. I Cell death was assessed using propidium iodide (PI) staining in the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with the indicated doses of RSL3 for 24 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns no significance. J, K Migration (J) and cloning formation (K) abilities of the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells) for 6 h. Mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns no significance. L Lipid peroxidation of the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells) for 6 h. Mean ± SD, n = 3. **P < 0.01, ****P < 0.0001. ns no significance. M Cell death was assessed using propidium iodide (PI) staining in the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells) in the presence or absence of MitoQH2 (2.5 μM) for 24 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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