glutathione  (Chem Impex International)

Journal: Biology Direct

Article Title: Platelet-activating factor induces ferroptosis by binding to ATF3 and inhibiting the SLC7A11/GPX4 axis to suppress the progression of endometrial carcinoma

doi: 10.1186/s13062-025-00713-z

Figure Lengend Snippet: PAF induces ferroptosis via the ATF3/GPX4 and ATF3/SLC7A11 axes. (A) Heat map representing the significantly regulated transcription factors detected via RNA-seq analysis of AN3CA and HEC1B cells. (B , C) The mRNA expression levels of ATF3 and DDIT3 were in AN3CA and HEC1B cells treated with PAF (60 µM) for 48 h. (D , E) Protein expression of ATF3 in AN3CA and HEC1B cells treated with PAF (0, 20, 40, and 60 µM) for 48 h (D), and the quantifiable data are shown (E). (F) Confocal microscopy of AN3CA and HEC1B cells in the presence of PAF (60 µM). Cells were stained for ATF3 (green) and phalloidin (red). DAPI was used to visualize the nucleus (blue). Scale bar, 50 μm (left) or 100 μm (right). (G) The potential binding sites between ATF3 and GPX4/SLC7A11 were predicted by JASPAR. (H) Schematic diagram of ATF3 binding site-mutated SLC7A11 reporter vector (pro-SLC7A11-mut) and GPX4 reporter vector (pro-GPX4-mut). (I , J) Dual luciferase assay of the luciferase activity of AN3CA (I) and HEC1B (J) cells. (K) Immunoblotting analysis of the expression of GPX4 and SLC7A11 following ATF3 overexpression in AN3CA and HEC1B cells. (L) Immunoblotting analysis of the protein expression of SLC7A11, GPX4, and ATF3 after ATF3 silencing with siRNA coupled with PAF treatment in AN3CA cells. * p < 0.05, ** p < 0.01 and *** p < 0.001, ns . not significant

Article Snippet: SPR assays were performed by TopScience Co., Ltd. (Shanghai, China) to detect the binding between the recombinant ATF3 protein (TMPH-01164, TargetMol) and C16-PAF (74389-68-7, TargetMol), with the ATF3 protein and anti-ATF3 antibody combination used as the positive control.

Techniques: RNA Sequencing, Expressing, Confocal Microscopy, Staining, Binding Assay, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Over Expression

Journal: Biology Direct

Article Title: Platelet-activating factor induces ferroptosis by binding to ATF3 and inhibiting the SLC7A11/GPX4 axis to suppress the progression of endometrial carcinoma

doi: 10.1186/s13062-025-00713-z

Figure Lengend Snippet: PAF binds to ATF3 and reduces its ubiquitination. (A , B) Molecular docking indicates the binding details between PAF and ATF3. The surface representation of the protein residues ( A ) and 2D representation of the binding interaction of PAF and ATF3 ( B ) are depicted. (C , D) Surface plasmon resonance of the affinity of anti-ATF3 antibody ( C ) and PAF ( D ) for ATF3 protein. K D , dissociation constant. (E) WB analysis shows that PAF stabilized ATF3 across different temperature gradients in the CETSA in 293T cells. (F) WB analysis indicates that PAF promoted the resistance of ATF3 to pronase digestion in the DARTS assay in 293T cells. (G) CHX chase analysis of ATF3 protein expression after treatment with PAF in AN3CA and HEC1B cells. (H) WB analysis of ATF3 in ATF3 -overexpressing AN3CA and HEC1B cells. The cells were pretreated with PAF (60 µM) for 24 h and then treated with CHX and MG132 for 24 h. (I) Representative WB images demonstrate the ubiquitination of ATF3 in 293T cells co-transfected with ATF3-Flag, HA-Ub, and plasmids for 24 h. Cellular lysates were collected after 3 h of treatment with PAF, purified with a Flag-tag protein purification kit, and then subjected to WB with anti-HA and anti-ATF3

Article Snippet: SPR assays were performed by TopScience Co., Ltd. (Shanghai, China) to detect the binding between the recombinant ATF3 protein (TMPH-01164, TargetMol) and C16-PAF (74389-68-7, TargetMol), with the ATF3 protein and anti-ATF3 antibody combination used as the positive control.

Techniques: Ubiquitin Proteomics, Binding Assay, SPR Assay, Expressing, Transfection, Purification, FLAG-tag, Protein Purification

Journal: Biology Direct

Article Title: Platelet-activating factor induces ferroptosis by binding to ATF3 and inhibiting the SLC7A11/GPX4 axis to suppress the progression of endometrial carcinoma

doi: 10.1186/s13062-025-00713-z

Figure Lengend Snippet: Schematic overview of the present study. Treatment with PAF attenuates EC cell proliferation by inducing ferroptosis. Mechanistically, PAF interacts with ATF3, enhancing its stability through deubiquitination and consequently suppressing the expression of the ferroptosis-related proteins SLC7A11 and GPX4

Article Snippet: SPR assays were performed by TopScience Co., Ltd. (Shanghai, China) to detect the binding between the recombinant ATF3 protein (TMPH-01164, TargetMol) and C16-PAF (74389-68-7, TargetMol), with the ATF3 protein and anti-ATF3 antibody combination used as the positive control.

Techniques: Expressing