Journal: Cell Death Discovery

Article Title: Rottlerin triggers dual degradation of SLC7A11 and GPX4 to drive ferroptosis and chemosensitization in hepatocellular carcinoma

doi: 10.1038/s41420-026-02942-1

Figure Lengend Snippet: A HLE and LM3 cells were treated with 5 μM rottlerin for 0, 6, 12, and 24 h. The protein levels of GPX4 and SLC7A11 were detected by Western blot, with Vinculin as a loading control. B HLE and LM3 cells were pretreated with 5 μM rottlerin for 4 h, followed by the addition of 20 μM CQ or 10 μM MG132 for 6 h. GPX4 and SLC7A11 protein levels were detected by Western blot. C LM3 cells were pretreated with 5 μM rottlerin for 3 h, then treated with 100 μg/mL CHX for the indicated times. Cells were harvested and analyzed by Western blot. D Control (shCtrl) and PKCδ-knockdown (sh#1, sh#2) LM3 cells were treated with 5 μM rottlerin for 0, 6, 12, and 24 h. The protein levels of GPX4 and SLC7A11 were detected by Western blot. E HCC cells transfected with Flag-GPX4 or Flag-SLC7A11 were treated with or without 5 μM rottlerin for 48 h, then the cells were collected for PI staining and analyzed by flow cytometry. The representative images (Top) and cell death rates (Bottom) were displayed. Data were represented as mean ± SD ( n = 3) and analyzed using one-way ANOVA, *** P < 0.001. F HLE cells were transfected with the indicated plasmids for 24 h and then treated with or without 5 μM rottlerin for 24 h. Cells were lysed for immunoprecipitation with anti-Flag antibodies, followed by Western blot with the specified antibodies. G HLE cells were pretreated with or without 10 μM Wu-5 (USP10 inhibitor), 10 μM Degrasyn (USP9x, USP5, USP14 and UCH37 inhibitor), 10 μM BAP1-IN-1 (BAP1 inhibitor), 10 μM LS-102 (SYVN1 inhibitor), 5 μM USP8-IN-3 (USP7/USP8 inhibitor), 5 μM USP7/USP47 inhibitor (USP7/USP47 inhibitor), 25 μM Heclin (HECT inhibitor), 20 μM C25-140 (TRAF6 inhibitor), 20 μM IU1 (USP14 inhibitor), and 5 μM b-AP15 (UCHL5/USP14 inhibitor) for 1 h, before being treated with or without 5 μM rottlerin for 24 h. Western blots were performed with the indicated antibodies. H HLE cells were transfected with the indicated plasmids for 24 h and then pretreated with or without 10 μM LS-102 (SYVN1 inhibitor), 5 μM b-AP15 (UCHL5 /USP14 inhibitor), and 5 μM USP8-IN-3 (USP7/USP8 inhibitor) for 1 h, before being treated with or without 5 μM rottlerin for 24 h. Cells were lysed for immunoprecipitation with anti-Flag antibodies, followed by Western blot with the specified antibodies.

Article Snippet: USP7/USP47 inhibitor (KKL Med, Cat# 1247825-37-1, USA), Heclin (KKL Med, Cat# 890605-54-6, USA), C25-140 (KKL Med, Cat# 1358099-18-9, USA), IU1 (KKL Med, Cat# 314245-33-5, USA), b-AP15 (KKL Med, Cat# 1009817-63-3, USA), Wu-5 (TargetMol, Cat# 2630378-05-9, China), Degrasyn (TargetMol, Cat# 856243-80-6, China), BAP1-IN-1 (TargetMol, Cat# 353495-21-3, China), LS-102 (Ambeed, Cat# A1340566, China) and USP8-IN-3 (KKL Med, Cat# 2477651-10-6, USA).

Techniques: Western Blot, Control, Knockdown, Transfection, Staining, Flow Cytometry, Immunoprecipitation