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: 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

Journal: PLOS One

Article Title: Transcriptome profiling and weighted gene co-expression network analysis reveal changes of hub genes and molecular pathways in rat lungs following deep hypothermic circulatory arrest

doi: 10.1371/journal.pone.0328887

Figure Lengend Snippet: (A) The results of HE staining in lung tissues between the two groups. (B-I) The analysis of IHC staining for hub genes in lung tissues was conducted between the two groups. The genes examined, in sequence, included FOS, FOSB, JUN, EGR1, ATF3, NR4A1, CCN1, and ZFP36. HE: hematoxylin and eosin; IHC: immunohistochemistry.

Article Snippet: Primary antibodies were then incubated overnight at 4 °C: FOS (Bioss, bs-0469R), FOSB (Bioss, bsm-52071R), Jun (Bioss, bs-0670R), EGR1 (Bioss, bs-1076R), ATF3 (Bioss, bs-0519R), NR4A1 (Bioss, bsm-52972R), CCN1 (Bioss, bs-1290R) and ZFP36 (Bioss, bs-20893R).

Techniques: Staining, Immunohistochemistry, Sequencing

Journal: Journal of Dental Sciences

Article Title: Fatostatin delayed lip sensory recovery after inferior alveolar nerve transection by inhibiting sterol regulatory element-binding protein 1

doi: 10.1016/j.jds.2025.04.024

Figure Lengend Snippet: Fatostatin impaired lip sensory recovery following inferior alveolar nerve transection in vivo . (A) Quantitative sensory testing using Von Frey filaments showed the sensory recovery of the lower lip in the control and fatostatin-treated groups, measured every 4 days for 40 days. The results show that fatostatin significantly delayed lip sensory recovery compared to the control group. (B) Quantitative real-time PCR analysis of gene expression in the trigeminal ganglia demonstrated that fatostatin treatment significantly decreased the mRNA levels of SREBP1, FASN, ACLY, ATF3, and NGF. (C) Western blot analysis confirmed that fatostatin treatment reduced the expression levels of SREBP1, ACLY, and ATF3 in the trigeminal ganglia compared to the control group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. SREBP1, sterol regulatory element-binding protein 1; FASN, fatty acid synthase; ACLY, ATP citrate lyase; ATF3, activating transcription factor 3; NGF, nerve growth factor.

Article Snippet: The used primary antibodies included SREBP1 (sc-13551, Santa Cruz, Dallas, TX, USA), ACLY (sc-517267, Santa Cruz), ATF3 (18665, Cell Signaling Technology, Boston, MA, USA), GAPDH (GB15002, Servicebio, Wuhan, China) After washing in TBST, membranes were incubated with the secondary antibody (ZSGB-BIO, Beijing, China), washed again in TBST, and visualized.

Techniques: In Vivo, Control, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Expressing, Binding Assay

Journal: Journal of Dental Sciences

Article Title: Fatostatin delayed lip sensory recovery after inferior alveolar nerve transection by inhibiting sterol regulatory element-binding protein 1

doi: 10.1016/j.jds.2025.04.024

Figure Lengend Snippet: Fatostatin inhibits axonal growth of primary trigeminal neurons in vitro . (A) Images of cultured primary neurons stained with NF200. The upper panels show the control group (left) and the fatostatin-treated group (right) at 20 × magnification. The lower panels show higher magnification (100 × ) of neurons in both groups. Scale bars in the upper panels indicate 100 μm, and in the lower panels, 20 μm. (B) Quantitative analysis of axonal length in the top 50 neurons, showing that fatostatin significantly reduced axonal growth compared to the control group. (C) Quantitative PCR analysis of key genes related to lipid metabolism and axonal regeneration, including SREBP1, FASN, ACLY, ATF3, and NGF. The results show that fatostatin treatment significantly reduced the expression levels of these genes, with SREBP1 and FASN showing the most prominent changes. (D) Western blot analysis of SREBP1, ACLY, and ATF3 protein expression levels, demonstrating that fatostatin treatment decreased SREBP1, ACLY, and ATF3 protein levels compared to control. GAPDH was used as the loading control. ∗∗∗, P < 0.001, ∗∗∗∗, P < 0.0001. SREBP1, sterol regulatory element-binding protein 1; FASN, fatty acid synthase; ACLY, ATP citrate lyase; ATF3, activating transcription factor 3; NGF, nerve growth factor.

Article Snippet: The used primary antibodies included SREBP1 (sc-13551, Santa Cruz, Dallas, TX, USA), ACLY (sc-517267, Santa Cruz), ATF3 (18665, Cell Signaling Technology, Boston, MA, USA), GAPDH (GB15002, Servicebio, Wuhan, China) After washing in TBST, membranes were incubated with the secondary antibody (ZSGB-BIO, Beijing, China), washed again in TBST, and visualized.

Techniques: In Vitro, Cell Culture, Staining, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Binding Assay

Journal: Research

Article Title: Piezo1-Mediated Ferroptosis Delays Wound Healing in Aging Mice by Regulating the Transcriptional Activity of SLC7A11 through Activating Transcription Factor 3

doi: 10.34133/research.0718

Figure Lengend Snippet: Inhibition of the Piezo1 channel alleviates ferroptosis in HUVECs via a Ca 2+ -dependent mechanism. (A) Schematic of in vivo experiment. (B) Wound images during healing and schematic diagram of wound-healing process. (C) Quantitative data of relative wound area to that of day 0 of the 4 groups. (D and E) H&E and Masson staining images on days 3 and 7. (F to H) Representative IHC images of VEGF-A, ATF3, and CaMKII. (I to K) Representative IF images of GPX4, SLC7A11, and α-SMA. (L) Laser Doppler scanned images of subcutaneous vascular flow and blood supply on wounds at days 3 and 7. *** P < 0.001, ns = no significant. All data are performed in triplicate and at least 3 times.

Article Snippet: Cells were fixed and lysed in a gentle lysis buffer, and 90% of the lysate was incubated with ATF3-specific antibodies (from Beyotime, catalog no. P2171, China) and an immunoglobulin G (IgG) control (same supplier) for 24 h. Complexes were then pelleted, washed 3 times with lysis buffer, and denatured before being loaded onto a denaturing SDS-PAGE gel for analysis.

Techniques: Inhibition, In Vivo, Staining

Journal: Research

Article Title: Piezo1-Mediated Ferroptosis Delays Wound Healing in Aging Mice by Regulating the Transcriptional Activity of SLC7A11 through Activating Transcription Factor 3

doi: 10.34133/research.0718

Figure Lengend Snippet: Blockade of ATF3 protects against ferroptosis in erastin- and Doxo-treated HUVECs. (A) Schematic diagram of RNA sequencing design and sample preparation procedures. (B and C) GSEA shows ferroptosis and lipid metabolism in Doxo group and Doxo + si-Piezo1 group. (D) Venn diagram showing these overlapping genes between 2 clusters (Doxo versus Doxo + si-Piezo1 and Ferroptosis) to obtain the intersection of the 2 DEGs. (E and F) GO and KEGG enrichment analyses showing calcium-mediated signaling, lipid metabolic process, and ferroptosis associated with these DEGs. (G and H) Western blot analysis for n-ATF3 in HUVECs with different treatments. (I and O) Representative IF images and quantification analysis of ATF3. (J to M) Representative images of JC-1 staining, Mito Green, C11-BODIPY staining, and FerroOrange staining in HUVECs with different treatments. (N and P) Western blot analysis for ferroptosis biomarkers ACSL4 and GPX4 in HUVECs with different treatments. (Q) LDH release of HUVECs with different treatments for 24 h. (R) GSH/GSSG ratio was measured in HUVECs with different treatments. *** P < 0.001. All data are performed in triplicate and at least 3 times.

Article Snippet: Cells were fixed and lysed in a gentle lysis buffer, and 90% of the lysate was incubated with ATF3-specific antibodies (from Beyotime, catalog no. P2171, China) and an immunoglobulin G (IgG) control (same supplier) for 24 h. Complexes were then pelleted, washed 3 times with lysis buffer, and denatured before being loaded onto a denaturing SDS-PAGE gel for analysis.

Techniques: RNA Sequencing, Sample Prep, Western Blot, Staining

Journal: Research

Article Title: Piezo1-Mediated Ferroptosis Delays Wound Healing in Aging Mice by Regulating the Transcriptional Activity of SLC7A11 through Activating Transcription Factor 3

doi: 10.34133/research.0718

Figure Lengend Snippet: Activation of Piezo1 by Yoda1 facilitates CaMKII-ATF3 interaction in HUVECs. (A to C) Western blot analysis for n-ATF3, CaMKII, SLC7A11, and GPX4 in HUVECs. (D and E) Representative IF images and quantification analysis of CaMKII and ATF3. (F) Three-dimensional binding structure of CaMKII and ATF3 determined via molecular modeling and docking studies. (G) Close-up view of hydrogen bonding between CaMKII and ATF3. (H) The interaction between CaMKII and ATF3 in HUVECs was analyzed by Co-IP. (I) Schematic diagram of the effects of Doxo and EGTA on the binding between CaMKII and ATF3. *** P < 0.001. All data are performed in triplicate and at least 3 times.

Article Snippet: Cells were fixed and lysed in a gentle lysis buffer, and 90% of the lysate was incubated with ATF3-specific antibodies (from Beyotime, catalog no. P2171, China) and an immunoglobulin G (IgG) control (same supplier) for 24 h. Complexes were then pelleted, washed 3 times with lysis buffer, and denatured before being loaded onto a denaturing SDS-PAGE gel for analysis.

Techniques: Activation Assay, Western Blot, Binding Assay, Co-Immunoprecipitation Assay

Journal: Research

Article Title: Piezo1-Mediated Ferroptosis Delays Wound Healing in Aging Mice by Regulating the Transcriptional Activity of SLC7A11 through Activating Transcription Factor 3

doi: 10.34133/research.0718

Figure Lengend Snippet: ATF3 represses SLC7A11 expression by regulating system Xc − . (A and B) Western blot analysis for ATF3-OE in HUVECs. (C and D) Representative IF images and quantification analysis of ATF3. (E and F) Western blot analysis for n-ATF3 and ferroptosis biomarkers SLC7A11 and GPX4 in HUVECs with different treatments. (G and H) Western blot analysis for ferroptosis biomarkers SLC7A11 and GPX4 in HUVECs with different treatments. (I) GSH/GSSG ratio was measured in HUVECs with different treatments. (J) Predicted ATF3-binding sites in the promoter region of SLC7A11. (K) A schematic illustration depicts the ATF3 motif within the promoter region of the SLC7A11 locus. (L) Luciferase assays were performed in 293T cell. (M) Schematic presentation of a model whereby ATF3 represses SLC7A11 expression to suppress system Xc − , and thereby predisposes cells to a state prone to ferroptosis. (N) The chromatin immunoprecipitation sequencing data previously reported were reanalyzed ( GSM1917770 , ENCSR632DCH_2, GSM803508 , GSM803503 ). *** P < 0.001. All data are performed in triplicate and at least 3 times.

Article Snippet: Cells were fixed and lysed in a gentle lysis buffer, and 90% of the lysate was incubated with ATF3-specific antibodies (from Beyotime, catalog no. P2171, China) and an immunoglobulin G (IgG) control (same supplier) for 24 h. Complexes were then pelleted, washed 3 times with lysis buffer, and denatured before being loaded onto a denaturing SDS-PAGE gel for analysis.

Techniques: Expressing, Western Blot, Binding Assay, Luciferase, ChIP-sequencing

Journal: Research

Article Title: Piezo1-Mediated Ferroptosis Delays Wound Healing in Aging Mice by Regulating the Transcriptional Activity of SLC7A11 through Activating Transcription Factor 3

doi: 10.34133/research.0718

Figure Lengend Snippet: The down-expression of ATF3 inhibits ferroptosis through the regulation of the Xc − system. (A) Schematic of in vivo experiment. (B and C) Wound images during healing and schematic diagram of wound-healing process. (D) Quantitative data of relative wound area to that of day 0 of the 4 groups. (E and F) H&E and Masson staining images on days 3 and 7. (G) Representative IHC images of VEGF-A. (H to J) Representative IF images of SLC7A11, α-SMA, and 4-HNE. (K) Laser Doppler scanned images of subcutaneous vascular flow and blood supply on wounds at day 3 and 7. *** P < 0.001. All data are performed in triplicate and at least 3 times.

Article Snippet: Cells were fixed and lysed in a gentle lysis buffer, and 90% of the lysate was incubated with ATF3-specific antibodies (from Beyotime, catalog no. P2171, China) and an immunoglobulin G (IgG) control (same supplier) for 24 h. Complexes were then pelleted, washed 3 times with lysis buffer, and denatured before being loaded onto a denaturing SDS-PAGE gel for analysis.

Techniques: Expressing, In Vivo, Staining

Journal: Research

Article Title: Piezo1-Mediated Ferroptosis Delays Wound Healing in Aging Mice by Regulating the Transcriptional Activity of SLC7A11 through Activating Transcription Factor 3

doi: 10.34133/research.0718

Figure Lengend Snippet: The deficiency of ATF3 promotes wound healing and angiogenesis in aging mice. Piezo1 up-regulated by calcium ion influx triggers ATF3 nuclear translocation via activation of CaMKII, thus aggravating the accumulation of ROS and lipid peroxidation, and eventually leading to ferroptosis in senescent HUVECs.

Article Snippet: Cells were fixed and lysed in a gentle lysis buffer, and 90% of the lysate was incubated with ATF3-specific antibodies (from Beyotime, catalog no. P2171, China) and an immunoglobulin G (IgG) control (same supplier) for 24 h. Complexes were then pelleted, washed 3 times with lysis buffer, and denatured before being loaded onto a denaturing SDS-PAGE gel for analysis.

Techniques: Translocation Assay, Activation Assay