Journal: Clinical and Translational Medicine

Article Title: MUC1 drives ferroptosis resistance in ICC via Src‐mediated FSP1 deubiquitination and myristoylation

doi: 10.1002/ctm2.70495

Figure Lengend Snippet: MUC1 interacts with and activates Src kinase to regulate FSP1 expression. (A) MUC1 interactome identification in HuCCT1 cells: left panel shows Coomassie blue staining of immunoprecipitated proteins; right panel displays tabulated IP–MS results of MUC1‐associated proteins. (B) Co‐immunoprecipitation assays demonstrating interaction between MUC1 and Src in HuCCT1 cells. (C) In vitro GST pull‐down assay confirming direct binding between recombinant MUC1 and Src proteins. (D) Immunofluorescence co‐localisation analysis of MUC1 (green) and Src (red) in HuCCT1 cells. Representative images shown at 600× magnification; scale bar = 10 µm. (E) Western blot analysis of Src phosphorylation at Tyr419 in HuCCT1 cells with MUC1 manipulation. (F) Western blot analysis of FSP1 protein levels in HuCCT1 cells with MUC1 overexpression treated with or without Src inhibitors. (G) Ferrorrange staining for ferrous ion detection in HuCCT1 cells under indicated treatments. Representative images shown at 200× magnification; scale bar = 25 µm. (H) Lipid peroxidation levels measured by MDA assay. (I) Flow cytometric analysis of intracellular ROS levels using DCFH‐DA staining. (J) Representative images of subcutaneous tumours from different treatment groups: control, RSL3 treatment and RSL3 + dasatinib combination therapy. (K) Quantitative analysis of tumour weights across treatment groups. Data are shown as the means ± SD, the significant level was identified by * p < .05; ** p < .01; *** p < .001.

Article Snippet: Recombinant active Src kinase (Sino Biological, Beijing, China) was incubated with purified USP10 or NMT1 proteins in kinase buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2 mM DTT, 1 mM Na 3 VO 4 , 5 mM β‐glycerophosphate and 100 μM ATP) at 30°C for 30 min.

Techniques: Expressing, Staining, Immunoprecipitation, Protein-Protein interactions, In Vitro, Pull Down Assay, Binding Assay, Recombinant, Immunofluorescence, Western Blot, Phospho-proteomics, Over Expression, Multiple Displacement Amplification, Control

Journal: Clinical and Translational Medicine

Article Title: MUC1 drives ferroptosis resistance in ICC via Src‐mediated FSP1 deubiquitination and myristoylation

doi: 10.1002/ctm2.70495

Figure Lengend Snippet: Src phosphorylates USP10 to regulate FSP1 stability. (A) Co‐immunoprecipitation assays demonstrating interaction between Src and USP10 in HuCCT1 cells. (B) In vitro GST pull‐down assay confirming direct binding between recombinant Src and USP10 proteins. (C–F) Domain mapping experiments identifying critical interaction regions: (C) schematic of Src domain mutants, (D) GST pull‐down assay with Src mutants, (E) schematic of USP10 domain mutants, (F) GST pull‐down assay with USP10 mutants. (G) Phos‐tag SDS‐PAGE analysis of USP10 phosphorylation status following in vitro kinase reactions with purified Src, MUC1 and Src inhibitor. (H) Co‐immunoprecipitation assays examining Src–USP10 interaction in HuCCT1 cells with MUC1 knockdown.

Article Snippet: Recombinant active Src kinase (Sino Biological, Beijing, China) was incubated with purified USP10 or NMT1 proteins in kinase buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2 mM DTT, 1 mM Na 3 VO 4 , 5 mM β‐glycerophosphate and 100 μM ATP) at 30°C for 30 min.

Techniques: Immunoprecipitation, In Vitro, Pull Down Assay, Binding Assay, Recombinant, SDS Page, Phospho-proteomics, Purification, Knockdown