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, Analysis, Expressing, Confocal, Microscopy, Staining, Binding Assay, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Over Expression, Real-time Polymerase Chain Reaction, Immunofluorescence

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, Docking Assay, Binding Assay, SPR Assay, Analysis, Expressing, Transfection, Purification, FLAG-tag, Protein Purification, Positive Control, Co-Immunoprecipitation Assay

Journal: Molecular Medicine Reports

Article Title: Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells

doi: 10.3892/mmr.2021.12111

Figure Lengend Snippet: Effect of anoxia in the presence or absence of the IDO inhibitor 1-MT on ATF4, CHOP, ATF3, p-p53 and p53 levels. Representative western blots for the levels of (A) ATF4, (B) CHOP, (C) ATF3, (D) p-p53 and (E) p53. Semi-quantification of (F) ATF4, (G) CHOP, (H) ATF3, (I) p-p53 and (J) p53 protein levels. (K) p-p53/total p53 ratio. *P<0.05 vs. control; # P<0.05 vs. control with 1-MT; ^ P<0.05 vs. anoxia; & P<0.05 vs. anoxia with 1-MT. 1-MT, 1-DL-methyltryptophan; IDO, indoleamine 2,3-dioxygenase 1; ATF4, activating transcription factor 4; CHOP; C/EBP homologous protein; ATF4, activating transcription factor 3; p-, phosphorylated; OD, optical density.

Article Snippet: Primary antibodies were specific the following proteins: IDO (1:200; cat. no. sc-25809), GCN2K (1:100; cat. no. sc-374609) (both from Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K; 1:1,000; cat. no. ab75836; Abcam), eukaryotic translation initiation factor-2α (eIF2α; 1:100; cat. no. sc-133132; Santa Cruz Biotechnology, Inc.), p at Ser51 eIF2α (p-eIF2α; 1:1,000; cat. no. 9721; Cell Signaling Technology, Inc.), activating transcription factor 4 (ATF4; 1:500; cat. no. CSB-PA002272KA01HU), ATF3 (1:500; cat. no. CSB-PA020022) (both Cusabio Technology LLC), C/EBP homologous protein (CHOP; 1:1,000; cat. no. 5554), p53 (1:1,000; cat. no. 2524), p at Ser15 p53 (p-p53; 1:1,000; cat. no. 9284), Bax (1:1,000; cat. no. 5023) (all Cell Signaling Technology, Inc.), death receptor 5 (DR5; 1:500; cat. no. CSB-PA018500; Cusabio Technology LLC), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam), AhR (1:200; cat. no. sc-133088), cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1; 1:500; cat. no. sc-25304) (both Santa Cruz Biotechnology, Inc.) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells

doi: 10.3892/mmr.2021.12111

Figure Lengend Snippet: IDO-mediated anoxia-induced apoptosis and reoxygenation-induced ferroptosis molecular pathways. IDO-mediated anoxia-induced apoptotic molecular pathway is depicted on the left. IDO-mediated reoxygenation-induced ferroptotic molecular pathway is depicted on the right. AIMP3/p18, aminoacyl-tRNA synthetase-interacting multifunctional protein-3/p18; AhR, aryl-hydrocarbon receptor; ATF3, activating transcription factor 3; ATF4, activating transcription factor 4; ATM/ATR, ataxia-telangiectasia mutated/ataxia-telangiectasia and Rad3 related protein complex; CHOP, C/EBP homologous protein; CC3, cleaved caspase-3; CYP1A1, cytochrome P450 family 1 subfamily A polypeptide 1; DR5, death receptor 5; IDO, indoleamine 2,3-dioxygenase 1; Kyn, kynurenine; p-, phosphorylated; eIF2a, eukaryotic translation initiation factor-2α; GCN2K, general control nonderepressible-2 kinase; MRS, methionyl-tRNA synthetase; p53, p53; ROS, reactive oxygen species; Trp, tryptophan.

Article Snippet: Primary antibodies were specific the following proteins: IDO (1:200; cat. no. sc-25809), GCN2K (1:100; cat. no. sc-374609) (both from Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K; 1:1,000; cat. no. ab75836; Abcam), eukaryotic translation initiation factor-2α (eIF2α; 1:100; cat. no. sc-133132; Santa Cruz Biotechnology, Inc.), p at Ser51 eIF2α (p-eIF2α; 1:1,000; cat. no. 9721; Cell Signaling Technology, Inc.), activating transcription factor 4 (ATF4; 1:500; cat. no. CSB-PA002272KA01HU), ATF3 (1:500; cat. no. CSB-PA020022) (both Cusabio Technology LLC), C/EBP homologous protein (CHOP; 1:1,000; cat. no. 5554), p53 (1:1,000; cat. no. 2524), p at Ser15 p53 (p-p53; 1:1,000; cat. no. 9284), Bax (1:1,000; cat. no. 5023) (all Cell Signaling Technology, Inc.), death receptor 5 (DR5; 1:500; cat. no. CSB-PA018500; Cusabio Technology LLC), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam), AhR (1:200; cat. no. sc-133088), cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1; 1:500; cat. no. sc-25304) (both Santa Cruz Biotechnology, Inc.) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Control