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. 2023 Dec 7;14(12):800. doi: 10.1038/s41419-023-06333-7

Fig. 4. The level of ALOX5 affects the sensitivity of BCa cells to ferroptosis in vitro.

Fig. 4

A Intracellular total ROS levels were measured using the DCFH-DA probe in 5637 and T24 cells transfected with ALOXs siRNA. Scale bars, 100 μm. B The fluorescence intensity was detected using a fluorescence microplate reader. C BCa cells transfected with ALOXs siRNA were treated with RSL3 for 24 h, and the cell death ratio was detected using trypan blue at a concentration of 0.4%. D Representative images of BCa cells treated with RSL3 (1 μM) and specific inhibitors of ALOX5 (Zileuton, 40 μM) or ALOX12 (ML355, 4 μM) for 24 h. Scale bars, 100 μm. E BCa cells transfected with ALOX5 siRNA or control were treated with RSL3 (1 μM), and the lipid ROS level was measured using the C11-BODIPY probe. Scale bars, 50 μm. F The expression levels of ALOX5, ALOX12, and ALOX15 in UMUC3 and J82 cells after transfection with ALOXs overexpression plasmids. G, H BCa cells were transfected with ALOXs overexpression plasmids for 24 h and then treated with RSL3 (2 μM) for 24 h. The intracellular total ROS levels were measured using the DCFH-DA probe. The fluorescence intensity was assessed using a fluorescence microplate reader. Scale bars, 100 μm. I, J Viability (%) of BCa cells (UMUC3 and J82) after transfection with ALOXs overexpression plasmids for 24 h, and then treated with RSL3 (0.5, 1, 2, 3 μM) for 48 h. n.s., represent no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The data are represented as mean ± SD of three independent assays.