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. 2023 Jul 22;4(4):e337. doi: 10.1002/mco2.337

FIGURE 3.

FIGURE 3

Overexpression of ubiquitin‐specific protease 5 (USP5) inhibits ferroptosis. (A) The effect of USP5 on ferroptosis based on gene set enrichment analysis (GSEA) (“WikiPathways [WP] Ferroptosis,” “Hallmark reactive oxygen species pathway,” and “Gene Ontology Biological Process [GOBP] iron ion transport”). (B) Representative phase‐contrast images of LM3 cells stably overexpressing USP5 treated with RSL‐3 (10 μM, 24 h). (C) Analysis of the responses of LM3 cells overexpressing USP5 to ferrostatin (Fer‐1) (10 μM, 24 h) and RSL‐3 (10 μM, 24 h) was done using cell counting kit‐8 (CCK8) assay. (D) The expression level of lymphoid‐specific helicase (LSH) and ferroptosis‐related proteins are detected using western blot in LM3 cells stably overexpressing USP5. (E and F) The levels of total GSH (E) and lipid ROS (F) in LM3 cells stably overexpressing USP5 were analyzed. (G) Western blot was used to test the ferroptosis‐related proteins in xenograft tumors from LM3 cells overexpressing USP5. (H) The response of HepG2 cells transfected with USP5‐WT or USP5‐C335A to ferrostatin (10 μM, 24 h) and RSL‐3 (10 μM, 24 h) was analyzed by CCK8 assays. (I) The levels of total glutathione (GSH) in HepG2 cells transfected with USP5‐WT or USP5‐C335A were analyzed. Ns, nonsignificant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001.