Journal: Redox Biology

Article Title: Redox-triggered USP18 confers cisplatin resistance in ovarian cancer by selectively activating a non-canonical FSP1-dependent ferroptosis escape pathway

doi: 10.1016/j.redox.2026.104179

Figure Lengend Snippet: USP18 stabilizes IGF2BP2 by direct binding and deubiquitination, and IGF2BP2 regulates FSP1 via m6A methylation. a Silver staining of protein bands co-precipitated with USP18 from total protein extracts of A2780 cells transiently transfected with control Flag or USP18-Flag plasmids. b List of RNA-regulatory genes highly expressed in ovarian cancer identified by IP-MS of USP18-overexpressing A2780 cells. c 3D molecular docking model of USP18-IGF2BP2 interaction. d Co-IP assay detecting USP18-IGF2BP2 binding in USP18-overexpressing cells using Flag antibody. e Protein expression levels of USP18 and IGF2BP2 in OVCAR-3 cells overexpressing USP18 or CAOV3 cells with USP18 knockdown. f Representative images and quantification of IGF2BP2-positive rate among USP18-positive tumor cells in normal ovarian and ovarian cancer tissues. Scale bar: 100 μm g Representative images and quantification of IGF2BP2-positive rate among USP18-positive tumor cells in cisplatin-sensitive and -resistant ovarian cancer tissues. Scale bar: 100 μm h Representative immunofluorescence images (left) and co-localization analysis (right) in CAOV3 cells. Blue: DAPI; Red: USP18; Green: IGF2BP2. Scale bar: 30 μm. i Correlation analysis between IGF2BP2 and FSP1 expression in TCGA database. Analysis was performed via the GEPIA platform. j Survival analysis of patients with differential IGF2BP2 expression using data from TCGA. Analysis was performed via the KM-plotter platform. k Western blot analysis of IGF2BP2 protein stability in USP18-overexpressing OVCAR-3 cells and control cells following cycloheximide (CHX, 100 μg/mL) treatment. l Western blot analysis of IGF2BP2 levels in control (siNC) and USP18-knockdown (siUSP18) CAOV3 cells treated with or without MG132(20 μM). m Schematic of IGF2BP2 domain truncations (top). Flag-IP and Western blot analysis of 293T cells co-transfected with Myc-USP18 and Flag-tagged IGF2BP2 truncation mutants (bottom). n Polyubiquitination analysis of IGF2BP2 in HEK293T cells co-transfected with Flag-IGF2BP2, Myc-USP18, and various HA-tagged ubiquitin mutants (Lys6, 11, 27, 29, 33, 48 or 63). o-p FSP1 mRNA stability assay: qPCR analysis of FSP1 mRNA levels at indicated time points after actinomycin D (5 μg/mL) treatment in OVCAR-3 cells overexpressing USP18 (o) or OVCAR-3 cells overexpressing IGF2BP2 (p). q Western blot analysis of IGF2BP2 and FSP1 protein expression in ovarian cancer cell lines. r RT-qPCR analysis of FSP1 mRNA expression in OVCAR-3 cells overexpressing USP18 or CAOV3 cells with USP18 knockdown. s FSP1 protein expression levels in OVCAR-3 cells overexpressing IGF2BP2 or CAOV3 cells with IGF2BP2 knockdown. t RNA Immunoprecipitation (RIP) assay in CAOV3 cells detecting IGF2BP2-FSP1 mRNA interaction by RT-qPCR. u Methylated RIP-qPCR (MeRIP-qPCR) analysis of m6A modification levels on FSP1 mRNA in CAOV3 cells. Data presented as mean ± SD. Significance calculated by unpaired two-tailed t -test (r, t, u), while two-way ANOVA was used for comparisons across multiple factors (o, p). ∗P < 0.05; ∗∗∗∗P < 0.0001.

Article Snippet: 293T cells were co-transfected with IGF2BP2-Flag, USP18-myc-his, and HA-tagged plasmids containing different ubiquitination sites, followed by treatment with 20 μM MG132 for 8 h. Cells were harvested and lysed in IP lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitor cocktail.

Techniques: Binding Assay, Methylation, Silver Staining, Transfection, Control, Protein-Protein interactions, Co-Immunoprecipitation Assay, Expressing, Knockdown, Immunofluorescence, Western Blot, Ubiquitin Proteomics, Stability Assay, Quantitative RT-PCR, RNA Immunoprecipitation, Modification, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: circOMA1 delivered by exosomes regulates DRD2-mediated prolactinoma resistance

doi: 10.3892/ijmm.2026.5812

Figure Lengend Snippet: circOMA1 promotes ubiquitin-mediated degradation of DRD2 and induces CAB resistance in MMQ cells by upregulating KBTBD7. (A) Western blot detection of DRD2 protein in circNC and circOM cells (top) with quantification (bottom). (B) DRD2 protein levels in circNC and circOM cells were measured by western blotting after treatment with 100 μ g/ml CHX for varying lengths of time. (C) Western blot analysis of DRD2 in circOM cells at the indicated time points after CHX treatment with or without treatment with 20 μ M MG132. (D) Co-IP analysis of DRD2 ubiquitination in circNC and circOM cells (green arrow indicates DRD2). (E) Western blot and densitometric analysis of KBTBD7 expression in circNC and circOM cells. (F) Representative immunofluorescence images showing distribution and co-localisation of DRD2 and KBTBD7 in MMQ cells; the line profile at right indicates co-localisation. Scale bar, 10 μ m. (G) Co-IP demonstrating the interaction between DRD2 and KBTBD7 in MMQ cells. (H) Immunoblot analysis of KBTBD7, DRD2 and PRL after circOMA1 knockdown for 72 h in circOM cells. (I) Immunoblot detection of DRD2 in circOM cells after KBTBD7 knockout by CRISPR/Cas9. (J) CCK-8 determination of circOM cell sensitivity to CAB after KBTBD7 knockdown (R 2 =0.9240). (K) Western blot detection of KBTBD7 and DRD2 in PRL-PitNET-S (n=4) and PRL-PitNET-R (n=6) tissues. (L) Representative immunofluorescence images showing distribution and co-localisation of KBTBD7 and DRD2 in PRL-PitNET tissues. Scale bar, 10 μ m. Data are presented as the mean ± SEM of at least three repeats. Differences between groups were compared using a unpaired Student's t-test. * P<0.05 and ** P<0.01. CAB, cabergoline; NC, negative control; circ, circular RNA; PRL-PitNET, prolactin-secreting pituitary neuroendocrine tumour; PRL-PitNET-S, PRL-PitNET-sensitive; PRL-PitNET-R, PRL-PitNET-resistant; CHX, cycloheximide; MG132, proteasome inhibitor; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; Co-IP, co-immunoprecipitation; circ, circular RNA; DRD2, dopamine D2 receptor; KBTBD7, Kelch-repeat and BTB domain-containing protein 7.

Article Snippet: CHX (a protein synthesis inhibitor) and MG132 (a proteasome inhibitor) were purchased from MedChemExpress. circOM, circNC or MMQ cells were treated with 100 μ g/ml CHX alone or combined with 20 μ M MG132 for 0, 4, 8, or 12 h, after which, western blotting was performed.

Techniques: Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunofluorescence, Knockdown, Knock-Out, CRISPR, CCK-8 Assay, Negative Control, Immunoprecipitation