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༎HPGD promotes the binding of RNA-binding protein (RBP) <t>U2AF2</t> 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
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༎HPGD promotes the binding of RNA-binding protein (RBP) <t>U2AF2</t> 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
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༎HPGD promotes the binding of RNA-binding protein (RBP) <t>U2AF2</t> 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
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༎HPGD promotes the binding of RNA-binding protein (RBP) <t>U2AF2</t> 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
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༎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

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