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: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: ATF3 is localized at vascular endothelial cells (ECs) in the developing mouse retina (A) Retinal section staining of IB4 (green), ERG (magenta), and ATF3 (red) in WT mice at P5. Scale bars: 50 μm. (B) Schematic illustration of fluorescence-activated cell sorting (FACS) of mouse retinal ECs. (C) RT-qPCR analysis of mRNA in WT murine retinal non-EC (CD45-/CD31- - ) and EC (CD45-/CD31+) at P5. Error bars represent mean ± SEM. ∗ p < 0.05. See also Figures S1 and .

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Staining, Fluorescence, FACS, Quantitative RT-PCR

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Deficiency of ATF3 upregulated by VEGFA inhibits angiogenesis in vitro (A and B) RT-qPCR analysis of ATF3 mRNA in (A) HUVECs and (B) HRMECs stimulated with VEGFA (0, 10, 20, 40, and 80 ng/mL) for 3 h after serum starvation for 6 h. (C) HRMECs were stimulated with VEGFA (20 ng/mL) for 3 h after serum starvation for 6 h and stained with ATF3 (green) and DAPI (blue). Scale bars: 50 μm. (D) Tube formation assay on Matrigel using HRMECs transfected with negative control siRNAs (siNC) or ATF3 siRNAs (si ATF3 ). The vascular density (left) and vascular length density (right) were measured using the ImageJ Vessel Analysis plugin. (E) Tube formation assay on Matrigel using ATF3 -overexpressed ( ATF3 OE) HUVECs infected with lentiviral vectors of human ATF3 . The vascular density (left) and vascular length density (right) were measured using the ImageJ Vessel Analysis plugin. Scale bars: 50 μm. Error bars represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figures S3 and .

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: In Vitro, Quantitative RT-PCR, Staining, Tube Formation Assay, Transfection, Negative Control, Infection

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Endothelial ATF3 is required for postnatal retinal angiogenesis in mice (A) Schematic illustration of tamoxifen administration for the generation of Cdh5-Cre Atf3 fl/fl ( Atf3 iECKO) mice. (B) Retinal whole-mount staining of PECAM1 in Atf3 fl/fl (control) and Atf3 iECKO mice at P5. (C) Comparison of vascular progression lengths (control, n = 15 eyes; Atf3 iECKO, n = 12 eyes). Scale bars: 500 μm. (D) Retinal whole-mount staining of IB4 (green), ESM1 (red), and ERG (white) in control and Atf3 iECKO mice at P5. (E) Quantification of the proportion of the ESM1+ area relative to the ERG1 area in the vascular front (control, n = 7 eyes; Atf3 iECKO, n = 8 eyes). (F) Retinal whole-mount staining of PECAM1 (green), ERG (blue), ki67 (red), and ERG (white) in control and Atf3 iECKO mice at P5. Red color channel on the images was altered. (G) Number of ki67+/ERG+ cells/FOV in the vascular front (control, n = 10 eyes; Atf3 iECKO, n = 13 eyes). Scale bars: 100 μm. Error bars represent mean ± SEM. ∗∗∗ p < 0.001. See also Figure S5 .

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Staining, Control, Comparison

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: ATF3 expression is upregulated in endothelial cells of the OIR model (A) Schematic illustration of the mouse oxygen-induced retinopathy (OIR) model. (B) RT-qPCR analysis of ATF3 mRNA in normoxic (control) and OIR retinas at P17. Error bars represent mean ± SEM. ∗ p < 0.05. (C) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR-WT mice at P12. (D) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR-WT mice at P17. (E) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR WT mice at P21. Scale bars: 100 μm. See also Figures S6 and .

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Expressing, Quantitative RT-PCR, Control, Staining

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Endothelial ATF3 deletion inhibits vascular remodeling of OIR retinas (A) Schematic illustration of the mouse OIR model and tamoxifen administration. (B–D) Retinal whole-mount staining of IB4 in Atf3 fl/fl (control) and Cdh5-Cre Atf3 fl/fl ( Atf3 iECKO) OIR mice at (B) P12, (C) P17, and (D) P21. (E–I) Quantification of the avascular area at (E) P12, (F) P17, and (G) P21, and neovascular tuft (NVT) areas at (H) P17 and (I) P21 (P12: control, n = 12 eyes; Atf3 iECKO, n = 13 eyes) (P17: control, n = 7 eyes; Atf3 iECKO, n = 8 eyes) (P21: control, n = 10 eyes; Atf3 iECKO, n = 11 eyes). Scale bars: 500 μm. Error bars represent mean ± SEM. ∗∗ p < 0.01.

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Staining, Control

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: scRNA-seq of retinal vascular ECs from the OIR mice (A) Schematic illustration of the scRNA-seq mouse OIR model. (B) Uniform Manifold Projection (UMAP) of CD45-/CD31+ cells from mouse retinas in OIR at P17. (B′) UMAP colored for expression of ATF3 . (C) Gene Ontology (GO) analysis of highly expressed genes in ATF3 -positive ECs.

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Expressing

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: RNA-seq analysis of HRMECs transfected with negative control or ATF3 siRNAs (A) Principal component analysis of HRMECs transfected with Silencer Select negative control siRNA (siNC) or ATF3 siRNA (siATF3). PC1, the first principal component explains 79% of the variance; PC2, the second principal component explains 9% of the variance. n = 4 replicates. (B) Heatmap highlighting differentially expressed top 20 genes between ATF3 KD HRMECs and control HRMECs. (C) Volcano plot highlighting differentially expressed top 20 genes in ATF3 KD HRMECs compared with control HRMECs. (D) Protein–protein interaction network of factors involved in angiogenesis that are commonly downregulated in ATF3 -negative ECs from OIR mice and ATF3 KD HRMECs. Red-colored factors are related to the VEGFA-VEGFR2 signaling pathway.

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: RNA Sequencing Assay, Transfection, Negative Control, Control

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Factors commonly downregulated in ATF3 -negative ECs from OIR mice and ATF3 KD HRMECs

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Over Expression, Inhibition, Tube Formation Assay, Expressing, Activity Assay, Migration

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Expression of angiogenesis-related factors in ATF3 knockdown or -overexpressed ECs (A) RT-qPCR analysis of angiogenesis-related factors in HRMECs transfected with negative control siRNAs (siNC) or ATF3 siRNAs (si ATF3 ). (B) RT-qPCR analysis of angiogenesis-related factors in control or ATF3- overexpressed ( ATF3 OE) HUVECs infected with lentiviral vectors of human ATF3 . Error bars represent mean ± SEM. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Transfection, Negative Control, Control, Infection

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet:

Article Snippet: Stealth siRNA for human ATF3 , Invitrogen , Cat#: 1299001.

Techniques: Recombinant, Transfection, Isolation, Expressing, Negative Control, Software

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: ATF3 is localized at vascular endothelial cells (ECs) in the developing mouse retina (A) Retinal section staining of IB4 (green), ERG (magenta), and ATF3 (red) in WT mice at P5. Scale bars: 50 μm. (B) Schematic illustration of fluorescence-activated cell sorting (FACS) of mouse retinal ECs. (C) RT-qPCR analysis of mRNA in WT murine retinal non-EC (CD45-/CD31- - ) and EC (CD45-/CD31+) at P5. Error bars represent mean ± SEM. ∗ p < 0.05. See also Figures S1 and .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Staining, Fluorescence, FACS, Quantitative RT-PCR

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Deficiency of ATF3 upregulated by VEGFA inhibits angiogenesis in vitro (A and B) RT-qPCR analysis of ATF3 mRNA in (A) HUVECs and (B) HRMECs stimulated with VEGFA (0, 10, 20, 40, and 80 ng/mL) for 3 h after serum starvation for 6 h. (C) HRMECs were stimulated with VEGFA (20 ng/mL) for 3 h after serum starvation for 6 h and stained with ATF3 (green) and DAPI (blue). Scale bars: 50 μm. (D) Tube formation assay on Matrigel using HRMECs transfected with negative control siRNAs (siNC) or ATF3 siRNAs (si ATF3 ). The vascular density (left) and vascular length density (right) were measured using the ImageJ Vessel Analysis plugin. (E) Tube formation assay on Matrigel using ATF3 -overexpressed ( ATF3 OE) HUVECs infected with lentiviral vectors of human ATF3 . The vascular density (left) and vascular length density (right) were measured using the ImageJ Vessel Analysis plugin. Scale bars: 50 μm. Error bars represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figures S3 and .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: In Vitro, Quantitative RT-PCR, Staining, Tube Formation Assay, Transfection, Negative Control, Infection

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Endothelial ATF3 is required for postnatal retinal angiogenesis in mice (A) Schematic illustration of tamoxifen administration for the generation of Cdh5-Cre Atf3 fl/fl ( Atf3 iECKO) mice. (B) Retinal whole-mount staining of PECAM1 in Atf3 fl/fl (control) and Atf3 iECKO mice at P5. (C) Comparison of vascular progression lengths (control, n = 15 eyes; Atf3 iECKO, n = 12 eyes). Scale bars: 500 μm. (D) Retinal whole-mount staining of IB4 (green), ESM1 (red), and ERG (white) in control and Atf3 iECKO mice at P5. (E) Quantification of the proportion of the ESM1+ area relative to the ERG1 area in the vascular front (control, n = 7 eyes; Atf3 iECKO, n = 8 eyes). (F) Retinal whole-mount staining of PECAM1 (green), ERG (blue), ki67 (red), and ERG (white) in control and Atf3 iECKO mice at P5. Red color channel on the images was altered. (G) Number of ki67+/ERG+ cells/FOV in the vascular front (control, n = 10 eyes; Atf3 iECKO, n = 13 eyes). Scale bars: 100 μm. Error bars represent mean ± SEM. ∗∗∗ p < 0.001. See also Figure S5 .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Staining, Control, Comparison

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: ATF3 expression is upregulated in endothelial cells of the OIR model (A) Schematic illustration of the mouse oxygen-induced retinopathy (OIR) model. (B) RT-qPCR analysis of ATF3 mRNA in normoxic (control) and OIR retinas at P17. Error bars represent mean ± SEM. ∗ p < 0.05. (C) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR-WT mice at P12. (D) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR-WT mice at P17. (E) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR WT mice at P21. Scale bars: 100 μm. See also Figures S6 and .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Staining

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Endothelial ATF3 deletion inhibits vascular remodeling of OIR retinas (A) Schematic illustration of the mouse OIR model and tamoxifen administration. (B–D) Retinal whole-mount staining of IB4 in Atf3 fl/fl (control) and Cdh5-Cre Atf3 fl/fl ( Atf3 iECKO) OIR mice at (B) P12, (C) P17, and (D) P21. (E–I) Quantification of the avascular area at (E) P12, (F) P17, and (G) P21, and neovascular tuft (NVT) areas at (H) P17 and (I) P21 (P12: control, n = 12 eyes; Atf3 iECKO, n = 13 eyes) (P17: control, n = 7 eyes; Atf3 iECKO, n = 8 eyes) (P21: control, n = 10 eyes; Atf3 iECKO, n = 11 eyes). Scale bars: 500 μm. Error bars represent mean ± SEM. ∗∗ p < 0.01.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Staining, Control

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: scRNA-seq of retinal vascular ECs from the OIR mice (A) Schematic illustration of the scRNA-seq mouse OIR model. (B) Uniform Manifold Projection (UMAP) of CD45-/CD31+ cells from mouse retinas in OIR at P17. (B′) UMAP colored for expression of ATF3 . (C) Gene Ontology (GO) analysis of highly expressed genes in ATF3 -positive ECs.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Expressing

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: RNA-seq analysis of HRMECs transfected with negative control or ATF3 siRNAs (A) Principal component analysis of HRMECs transfected with Silencer Select negative control siRNA (siNC) or ATF3 siRNA (siATF3). PC1, the first principal component explains 79% of the variance; PC2, the second principal component explains 9% of the variance. n = 4 replicates. (B) Heatmap highlighting differentially expressed top 20 genes between ATF3 KD HRMECs and control HRMECs. (C) Volcano plot highlighting differentially expressed top 20 genes in ATF3 KD HRMECs compared with control HRMECs. (D) Protein–protein interaction network of factors involved in angiogenesis that are commonly downregulated in ATF3 -negative ECs from OIR mice and ATF3 KD HRMECs. Red-colored factors are related to the VEGFA-VEGFR2 signaling pathway.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Transfection, Negative Control, Control

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Factors commonly downregulated in ATF3 -negative ECs from OIR mice and ATF3 KD HRMECs

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Over Expression, Inhibition, Tube Formation Assay, Expressing, Activity Assay, Migration

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Expression of angiogenesis-related factors in ATF3 knockdown or -overexpressed ECs (A) RT-qPCR analysis of angiogenesis-related factors in HRMECs transfected with negative control siRNAs (siNC) or ATF3 siRNAs (si ATF3 ). (B) RT-qPCR analysis of angiogenesis-related factors in control or ATF3- overexpressed ( ATF3 OE) HUVECs infected with lentiviral vectors of human ATF3 . Error bars represent mean ± SEM. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Transfection, Negative Control, Control, Infection

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet:

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Recombinant, Transfection, Isolation, Expressing, Negative Control, Software

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: ATF3 is localized at vascular endothelial cells (ECs) in the developing mouse retina (A) Retinal section staining of IB4 (green), ERG (magenta), and ATF3 (red) in WT mice at P5. Scale bars: 50 μm. (B) Schematic illustration of fluorescence-activated cell sorting (FACS) of mouse retinal ECs. (C) RT-qPCR analysis of mRNA in WT murine retinal non-EC (CD45-/CD31- - ) and EC (CD45-/CD31+) at P5. Error bars represent mean ± SEM. ∗ p < 0.05. See also Figures S1 and .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Staining, Fluorescence, FACS, Quantitative RT-PCR

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Deficiency of ATF3 upregulated by VEGFA inhibits angiogenesis in vitro (A and B) RT-qPCR analysis of ATF3 mRNA in (A) HUVECs and (B) HRMECs stimulated with VEGFA (0, 10, 20, 40, and 80 ng/mL) for 3 h after serum starvation for 6 h. (C) HRMECs were stimulated with VEGFA (20 ng/mL) for 3 h after serum starvation for 6 h and stained with ATF3 (green) and DAPI (blue). Scale bars: 50 μm. (D) Tube formation assay on Matrigel using HRMECs transfected with negative control siRNAs (siNC) or ATF3 siRNAs (si ATF3 ). The vascular density (left) and vascular length density (right) were measured using the ImageJ Vessel Analysis plugin. (E) Tube formation assay on Matrigel using ATF3 -overexpressed ( ATF3 OE) HUVECs infected with lentiviral vectors of human ATF3 . The vascular density (left) and vascular length density (right) were measured using the ImageJ Vessel Analysis plugin. Scale bars: 50 μm. Error bars represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figures S3 and .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: In Vitro, Quantitative RT-PCR, Staining, Tube Formation Assay, Transfection, Negative Control, Infection

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Endothelial ATF3 is required for postnatal retinal angiogenesis in mice (A) Schematic illustration of tamoxifen administration for the generation of Cdh5-Cre Atf3 fl/fl ( Atf3 iECKO) mice. (B) Retinal whole-mount staining of PECAM1 in Atf3 fl/fl (control) and Atf3 iECKO mice at P5. (C) Comparison of vascular progression lengths (control, n = 15 eyes; Atf3 iECKO, n = 12 eyes). Scale bars: 500 μm. (D) Retinal whole-mount staining of IB4 (green), ESM1 (red), and ERG (white) in control and Atf3 iECKO mice at P5. (E) Quantification of the proportion of the ESM1+ area relative to the ERG1 area in the vascular front (control, n = 7 eyes; Atf3 iECKO, n = 8 eyes). (F) Retinal whole-mount staining of PECAM1 (green), ERG (blue), ki67 (red), and ERG (white) in control and Atf3 iECKO mice at P5. Red color channel on the images was altered. (G) Number of ki67+/ERG+ cells/FOV in the vascular front (control, n = 10 eyes; Atf3 iECKO, n = 13 eyes). Scale bars: 100 μm. Error bars represent mean ± SEM. ∗∗∗ p < 0.001. See also Figure S5 .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Staining, Control, Comparison

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: ATF3 expression is upregulated in endothelial cells of the OIR model (A) Schematic illustration of the mouse oxygen-induced retinopathy (OIR) model. (B) RT-qPCR analysis of ATF3 mRNA in normoxic (control) and OIR retinas at P17. Error bars represent mean ± SEM. ∗ p < 0.05. (C) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR-WT mice at P12. (D) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR-WT mice at P17. (E) Retinal whole-mount staining of IB4 (green), ATF3 (red), and ERG (white) in control and OIR WT mice at P21. Scale bars: 100 μm. See also Figures S6 and .

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Staining

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Endothelial ATF3 deletion inhibits vascular remodeling of OIR retinas (A) Schematic illustration of the mouse OIR model and tamoxifen administration. (B–D) Retinal whole-mount staining of IB4 in Atf3 fl/fl (control) and Cdh5-Cre Atf3 fl/fl ( Atf3 iECKO) OIR mice at (B) P12, (C) P17, and (D) P21. (E–I) Quantification of the avascular area at (E) P12, (F) P17, and (G) P21, and neovascular tuft (NVT) areas at (H) P17 and (I) P21 (P12: control, n = 12 eyes; Atf3 iECKO, n = 13 eyes) (P17: control, n = 7 eyes; Atf3 iECKO, n = 8 eyes) (P21: control, n = 10 eyes; Atf3 iECKO, n = 11 eyes). Scale bars: 500 μm. Error bars represent mean ± SEM. ∗∗ p < 0.01.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Staining, Control

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: scRNA-seq of retinal vascular ECs from the OIR mice (A) Schematic illustration of the scRNA-seq mouse OIR model. (B) Uniform Manifold Projection (UMAP) of CD45-/CD31+ cells from mouse retinas in OIR at P17. (B′) UMAP colored for expression of ATF3 . (C) Gene Ontology (GO) analysis of highly expressed genes in ATF3 -positive ECs.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Expressing

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: RNA-seq analysis of HRMECs transfected with negative control or ATF3 siRNAs (A) Principal component analysis of HRMECs transfected with Silencer Select negative control siRNA (siNC) or ATF3 siRNA (siATF3). PC1, the first principal component explains 79% of the variance; PC2, the second principal component explains 9% of the variance. n = 4 replicates. (B) Heatmap highlighting differentially expressed top 20 genes between ATF3 KD HRMECs and control HRMECs. (C) Volcano plot highlighting differentially expressed top 20 genes in ATF3 KD HRMECs compared with control HRMECs. (D) Protein–protein interaction network of factors involved in angiogenesis that are commonly downregulated in ATF3 -negative ECs from OIR mice and ATF3 KD HRMECs. Red-colored factors are related to the VEGFA-VEGFR2 signaling pathway.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Transfection, Negative Control, Control

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Factors commonly downregulated in ATF3 -negative ECs from OIR mice and ATF3 KD HRMECs

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Over Expression, Inhibition, Tube Formation Assay, Expressing, Activity Assay, Migration

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet: Expression of angiogenesis-related factors in ATF3 knockdown or -overexpressed ECs (A) RT-qPCR analysis of angiogenesis-related factors in HRMECs transfected with negative control siRNAs (siNC) or ATF3 siRNAs (si ATF3 ). (B) RT-qPCR analysis of angiogenesis-related factors in control or ATF3- overexpressed ( ATF3 OE) HUVECs infected with lentiviral vectors of human ATF3 . Error bars represent mean ± SEM. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Transfection, Negative Control, Control, Infection

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet:

Article Snippet: HUVECs and HRMECs were cultured to 40%–60% confluence and transfected for 24 h with Stealth siRNA targeting human ATF3 (Invitrogen) and Stealth RNAi™ siRNA Negative Control (Invitrogen) or Silencer Select siRNAs targeting human ATF3 (Ambion, Austin, TX, USA) and Silencer Select Negative Control No. 2 siRNA (Invitrogen) by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) in accordance with the manufacturer’s instructions.

Techniques: Recombinant, Transfection, Isolation, Expressing, Negative Control, Software