Fig. 2: Knockdown of USP24 promotes ferroptosis in TNBC cells.

A, B MDA-MB-231 and MDA-MB-468 cells were stably transfected with two individual USP24 shRNAs or control shRNA. USP24 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 < 0.001, ****P < 0.0001. C Cell viability of the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with the indicated doses of RSL3 for 24 h. D Cell viability of the indicated MDA-MB-231 cell lines treated with the indicated doses of ML162, ML210, FIN56, and erastin for 24 h. E 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 < 0.05, ***P < 0.001, ****P < 0.0001. ns no significance. Migration (F) and cloning formation (G) 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 < 0.01, ***P < 0.001, ****P < 0.0001. ns no significance. H, I The representative phase-contrast and propidium iodide (PI) staining images of the indicated MDA-MB-231 three-dimensional spheroids treated with 0.5 μM RSL3 for 48 h. The relative PI fluorescence intensity was quantified as the ratio of red fluorescence intensity (PI) to blue fluorescence intensity (Hoechst 33342). Mean ± SD, n = 3. ***P < 0.001, ****P < 0.0001. ns no significance. J 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 < 0.0001. ns no significance. K Cell death was assessed using propidium iodide (PI) staining in the indicated MDA-MB-231 cell treated with 0.5 μM RSL3, in the presence of ferroptosis inhibitor ferrostain-1 (Fer-1, 2 μM) or apoptosis inhibitor Z-VAD-FMK (Z-AVD, 10 μM) for 24 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. ****P < 0.0001. ns no significance. L Lipid peroxidation of the indicated MDA-MB-231 cell lines treated with 0.5 μM RSL3, in the presence of ferroptosis inhibitor ferrostain-1 (Fer-1, 2 μM) or apoptosis inhibitor Z-VAD-FMK (Z-AVD, 10 μM) for 6 h. Mean ± SD, n = 3. ****P < 0.0001. ns no significance.