Journal: Molecular Cancer

Article Title: HPGD induces ferroptosis and autophagy to suppress esophageal squamous cell carcinoma through the LXA4–ERK1/2–U2AF2–TFRC axis

doi: 10.1186/s12943-025-02464-x

Figure Lengend Snippet: ༎HPGD promotes the binding of RNA-binding protein (RBP) U2AF2 to the TFRC promoter, which improves its transcription by suppressing ERK1/2 signaling activation. (A) Construction of truncated TFRC promoter plasmids and dual-luciferase reporter assay analysis of the TFRC promoter region after transfection into 293 T cells. (B) Biotin labeling of the TFRC promoter region obtained from the dual-luciferase reporter assay, followed by Lipofectamine transfection into 293T cells, ultrasonic lysis, biotin pulldown, and SDS-PAGE to analyze differential proteins via MS-MS. (C) Biotinylated promoter transfected into 293 T cells, followed by biotin pulldown and Western blot validation of promoter-binding proteins. (D) ChIP assay to validate U2AF2 binding to the TFRC promoter in ESCC cells. (E) A dual luciferase plasmid with a mutated U2AF2 binding site in the TFRC promoter was co-transfected with a U2AF2 overexpression plasmid into 293 T cells to measure luciferase activity. (F) RNA-RIP experiments were performed utilizing U2AF2 and IgG antibodies. Following the purification of RNA and protein, TFRC was detected through PCR, while U2AF2 was identified using immunoblotting. G-H. Western blot analysis of U2AF2 expression levels in cells after HPGD overexpression. I. ChIP assay analysis of TFRC promoter activity after treatment with the Erk1/2 activator EGF in control and HPGD overexpressing cells. J. FISH analysis with TFRC promoter probes and U2AF2 antibodies to detect colocalization. K-L. Western blot detection of interference efficiency after silencing U2AF2 with small interfering RNA. M-N. Western blot detection of TFRC expression after silencing U2AF2 in ESCC cells with HPGD overexpression. *: P < 0.05,** P < 0.01,*** P < 0.001, **** P < 0.0001

Article Snippet: Chromatin immunoprecipitation was conducted overnight at 4 °C with primary antibodies against U2AF2 (Proteintech, America) and 2 μl of normal rabbit IgG (provided in the ChIP Assay Kit).

Techniques: Binding Assay, RNA Binding Assay, Activation Assay, Luciferase, Reporter Assay, Transfection, Labeling, Lysis, SDS Page, Tandem Mass Spectroscopy, Western Blot, Biomarker Discovery, Plasmid Preparation, Over Expression, Activity Assay, Purification, Expressing, Control, Small Interfering RNA

Journal: bioRxiv

Article Title: U2AF regulates the translation and localization of nuclear-encoded mitochondrial mRNAs

doi: 10.1101/2024.09.18.613780

Figure Lengend Snippet: ( A ) Schematic for CRISPR-Cas9-mediated insertion of a C-terminal 3XFLAG tag into the U2AF1 gene locus. ( B ) Validation of U2AF1 endogenous 3XFLAG tagging by immunoprecipitation and western blot in whole cell lysates from parental (Ctl) and U2AF1 edited (3X) cell lines. ( C ) Volcano plot analysis of the cytoplasmic fraction from parental and U2AF1 wt/wt-3XFlag cell lines. Mitochondrial-associated proteins are highlighted in red. A dashed line denotes the threshold for statistical significance (adjusted p < 0.05). Data represent three biological replicates (n=3) for each sample group. ( D ) GSEA plot showing significant enrichment of the ‘Oxidative Phosphorylation’ category in U2AF1 wt/wt-3XFlag cells compared to the parental control, as determined by the KEGG gene set. ( E ) Super-Resolution TIRF microscopy visualization using DNA-PAINT in U2AF1 wt/wt-3XFlag cells. U2AF1 was labeled with an anti-FLAG antibody (red), and the outer mitochondrial membrane was identified with anti-TOMM22 (cyan). Insets show magnified areas highlighting the colocalization of U2AF1 with the mitochondrial outer membrane. Scale bar indicates 1 µm. ( F ) Schematic of the centrifugation method used to separate mitochondrial (mito)-enriched and cytoplasmic fractions from cell lysates. ( G ) Western blot analysis of U2AF1, U2AF2 and TOMM22 (mitochondrial marker) in cytoplasmic and mitochondrial fractions from U2AF1 wt/wt cells . ( H ) GSEA gene score plot of the nuclear-ER fraction from control and U2AF1 wt/wt-3XFlag cell lines. Translation-associated proteins are highlighted in blue. Data represent three biological replicates ( n = 3) for each sample group. ( I ) Western blot validation of U2AF1-interacting proteins GIGYF2, G3BP1, EIF3(J subunit) from the ER-nuc fraction of parental (Ctl) and U2AF1 edited (3X) cell lines.

Article Snippet: Primary antibodies were incubated in the blocking solution overnight at 4°C with gentle rocking, using the following dilutions: FLAG at 1:1000 (Sigma, F3165), U2AF2 at 1:1000 (Santa Cruz, sc-53942), EIF3J at 1:500 (Bethyl, A301-746A-M), GIGYF2 at 1:500 (Bethyl, A303-732A), and G3BP1 at 1:2000 (Invitrogen, PA5-29455).

Techniques: CRISPR, Biomarker Discovery, Immunoprecipitation, Western Blot, Phospho-proteomics, Control, Microscopy, Labeling, Membrane, Centrifugation, Marker