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: The Journal of Clinical Investigation

Article Title: Mechanisms and treatments of neuropathic itch in a mouse model of lymphoma

doi: 10.1172/JCI160807

Figure Lengend Snippet: ( A ) Double immunostaining for ATF3 and IB4 in cervical DRGs of naive and CTCL mice at day 60. Scale bar: 25 μm. ( B ) Double immunostaining of ATF3 and the neuronal markers NF200, P2X3, and CGRP in cervical DRGs from CTCL mice at day 60. Scale bar: 25 μm. ( C ) Percentage of ATF3 + , IB4 + , NF200 + , P2X3 + , and CGRP + neurons in the cervical DRG of CTCL mice at different time points. n = 3. Two-way ANOVA, F (12, 30) = 9.15, P < 0.0001. ( D ) Percentage of ATF3 colocalization with P2X3 + neurons in the cervical DRG of CTCL mice at different time points. n = 3. One-way ANOVA, F (3, 8) = 31.89, P < 0.0001. ( E ) Pie chart showing ATF3 colocalization with NF200 + , P2X3 + , and CGRP + neurons in the cervical DRG from a CTCL mouse on day 60. Data were collected from 3 animals. ( F ) Double immunostaining for CGRP and IB4 in the cervical spinal cord dorsal horn from CTCL mice at day 60. Arrowheads indicate the loss of IB4 + primary afferents in the IIi. Scale bar: 100 μm. Data are expressed as the mean ± SEM. One- or 2-way ANOVA with Bonferroni’s post hoc test, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 versus the naive group.

Article Snippet: The primary antibodies included anti-CGRP antibody (goat, 1:400, Bio-Rad, catalog 1720-9007), anti-ATF3 antibody (mouse, 1:400, Santa Cruz Biotechnology, catalog sc-81189), anti-NF200 antibody (rabbit, 1:5,000, Abcam, catalog ab8135), anti-P2X3 antibody (mouse, 1:500, Neuromics, catalog Gp10108), anti–β–tubulin III antibody (mouse, 1:500, R&D Systems, catalog MAB1195), anti-CD68 antibody (rat, 1:500, BioLegend, catalog 137011), anti–Gr-1 antibody (rat, 1:500, BioLegend, catalog 127623), anti-CD11c antibody (Armenian hamster, 1:500, BD Pharmingen, catalog 550283), cleaved caspase 3 antibody (rabbit, 1:400, Cell Signaling Technology, catalog 9661), and anti–IB4–Alexa Fluor 488 conjugate (Invitrogen, Thermo Fisher Scientific, 1:1,000, catalog 121411).

Techniques: Double Immunostaining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids

doi: 10.1016/j.jcmgh.2024.101366

Figure Lengend Snippet: Antibodies Used

Article Snippet: ATF3 , Rabbit , Novus, NBP1-85816 , 1:500.

Techniques:

Journal: Scientific Reports

Article Title: Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors

doi: 10.1038/s41598-018-35806-5

Figure Lengend Snippet: Usp9X inhibition causes Noxa increase and ER stress in MPNST cell lines. Ultrastructural analysis shows features of paraptosis. ( a , b ) ST88-14 cells were transfected for 24 h with either non-targeting (NT)-siRNA or Usp9X-siRNA ( a ) or treated with WP1130 at the concentration of 1.25 and 2.5 µM ( b ). Whole cell extracts were collected prior to Western blot analysis for ATF3, Noxa and ß-actin. Numbers shows protein quantification analyzed through ImageJ. N = 3. ( c – e ) Ultrastructural appearance of untreated control cells using TEM. ( f – h ) After treatment with WP1130 at the concentration of 2.5 µM ( f , g , h ) T265-2c cells showed extensive cytosolic vacuolization (f, red arrows) and swelling of ER (g, red arrowheads) and mitochondria (h, red arrows).

Article Snippet: Primary antibodies were obtained from the following sources: Usp9X (Cell Signaling, Danvers, MA #5751), Mcl-1 (Cell Signaling, Danvers, MA #5453), Noxa (Calbiochem, San Diego, CA #OP180), ATF4 (Cell Signaling, Danvers, MA #11815), ATF3 (Novus Biologicals, Littleton, CO #NBP1-85816), β-actin (Sigma, St. Louis, MO #A5316).

Techniques: Inhibition, Transfection, Concentration Assay, Western Blot, Control

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Rac is dominant over Rho in the initial transcriptome-wide response to ECM stiffness and preferentially represses ATF3. (A,B) Venn diagrams of differentially expressed genes in MEFs cultured with 10% FBS for 1 h on stiff versus soft hydrogels, stiff hydrogels with or without EHT1864, or stiff hydrogels with or without CT04. (C,D) The genes regulated by ECM and Rac in A,B were compared to GO gene lists for transcription factors (TFs), transcription co-regulators (co-reg) and histone modifiers. (E,F) Log 2 (fold change) values and adjusted P -values of the genes regulated by ECM stiffness and Rac and contained within the indicated GO terms above. (G) Serum-starved MEFs were plated on soft or stiff FN-coated hydrogels with 10% FBS for 1 h with DMSO (Ctrl), EHT1864 or CT04. Atf3 mRNA levels were quantified by RT-qPCR. The graph shows mean+s.e.m. ( n =4) with results normalized to the expression level on soft hydrogels. * P <0.05; *** P <0.001 (two-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Dose-dependent effects of ECM stiffness on Rac–GTP, Atf3 mRNA, cyclin D1 mRNA and S phase entry. Serum-starved MEFs were incubated in DMEM containing 10% FBS on FN-coated hydrogels of increasing stiffness (∼2, 8, 15 and 25 kPa). (A) Rac–GTP levels determined at 1 h and graphed relative to Rac activity on the softest hydrogel. Results show mean±s.e.m. ( n =4). (B,C) Atf3 and cyclin D1 mRNA levels determined after 9 h and graphed relative to the mRNA levels on the softest hydrogel. Results show mean±s.e.m. ( n =3). (D) The percentage of EdU-positive nuclei determined at 24 h and graphed relative to EdU incorporation on the softest hydrogel. Results show mean±s.d. ( n =3). Statistical significance for each panel was determined by one-way ANOVA; asterisks show the results of Dunnett's post-tests relative to the softest hydrogel. * P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Incubation, Activity Assay

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: ATF3 repression linked to stiffness-dependent cyclin D1 expression. (A) Serum-starved MEFs on soft or stiff FN-coated hydrogels in DMEM containing 10% FBS were treated with vehicle (DMSO) or EHT1864 for 9 h. Atf3 and cyclin D1 mRNA levels were determined from the same lysates and normalized to mRNA expression levels in cells on soft hydrogels. Results show mean±s.e.m. ( n =3). (B) MEFs were infected with adenoviruses encoding GFP (control) or Rac V12 , serum-starved, and cultured and analyzed as in panel A. Results show mean±s.e.m. ( n =3). (C) MEFs infected with adenoviruses (Ad) encoding GFP (control) or ATF3 were serum-starved, incubated on FN-coated hydrogels with 10% FBS for 15 h, and analyzed by immunoblotting for cyclin D1 and ATF3 with GAPDH as the loading control. (D) Quantification of the immunoblot results in C. The graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal on the soft hydrogels ( n =3). (E,F) Serum-starved ROSA and ATF3 KO MEFs were incubated on stiff FN-coated hydrogels with DMSO (Ctrl) or EHT1864 for 9 h. Lysates were analyzed for the levels of ATF3 and cyclin D1 by immunoblotting. GAPDH was used as the loading control. Panel E shows results from ROSA clone R12 and ATF3 KO clone 1-20, and panel F shows quantification of the combined results from ROSA clones R11, R12 and R15 and ATF3 KO clones 1-20 and 1-29. Data were accrued from four independent experiments, and the graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal in the ROSA control. (G) S phase entry was analyzed by EdU incorporation in ROSA clones (R3, R11, R12 and R15) and ATF3 KO clones (1-20, 1-29, 1-44, 1-48 and 1-49) after serum starvation and incubation on stiff FN-coated hydrogels in DMEM containing 10% FBS for 24 h with DMSO (Ctrl) or EHT1864. Results show mean+s.d. n =7 for the ROSA clones and n =8 for the ATF3 KO clones. (H) Model showing that cyclin D1 is regulated by ECM stiffness and Rac through ATF3. * P <0.05; ** P <0.01; **** P <0.0001 (D, two-tailed unpaired t -tests; F and G, one-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Western Blot, Clone Assay, Two Tailed Test, One-tailed Test

Journal: Brain structure & function

Article Title: Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF

doi: 10.1007/s00429-023-02635-w

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: ATF3 Used for injured motoneuron identification , Recombinant protein corresponding to aa 1-103 in human ATF3 , Mouse/monoclonal , Novus Clone 1685 NBP2-34489 , AB_2786997 Recognizes the epitope: ASAIVPCLSPPGSL (Manufacturer’s information) Not present in uninjured motoneurons , 1:200.

Techniques: Comparison, Expressing, Recombinant