atf3 shrna lentiviral transduction particles  (Santa Cruz Biotechnology)

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

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: ATF3 is predicted as a favorable prognostic factor and is positively associated with GDF15 in bladder cancer. (A) Data of TCGA gained from the GEPIA database showed ATF3 expression levels across 31 kinds of tumor samples and paired normal tissues. The bar height represents the median expression of ATF3 in certain tumor types or normal tissues, as indicated. (B) Expression levels of ATF3 in the TCGA database from 28 normal and 404 tumor groups, respectively. (C) Correlation between ATF3 expression levels and progression-free survival (P.F.S.) of bladder urothelial carcinoma patients generated from TCGA-BLCA database. (D) The co-expression of ATF3 with GDF15 in TCGA-BLCA database. (E) Kaplan-Meier analysis of progression-free survival of TCGA-BLCA database, comparing subsets with double high expressions of ATF3 and GDF15 to those with double low expressions. Abbreviation: ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukemia; BLGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; and UCS: uterine carcinosarcoma. ∗, p < 0.05.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Generated

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of prognostic factors and ATF3 RNA expression in bladder cancer (N = 169).

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: RNA Expression, Biomarker Discovery, Expressing

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of prognostic factors and GDF15/ATF3 RNA expression in bladder cancer (N = 89).

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: RNA Expression, Biomarker Discovery, Expressing

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of ATF3 on cell growth and gene expression in bladder cancer cells. (A, left) The expressions of ATF3, GDF15, NDRG1, KAI-1, and β-actin of ectopic ATF3-overexpressed T24 (T24-ATF3) and mock-overexpressed T24 (T24-DNA) cells were determined by immunoblot assays. (A, right) Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin relative to the mock-control group (±SE; n = 3). Cell growth rate of T24-DNA and T24-ATF3 cells was determined by (B) Ki67 or (C) colony formation assays ( n = 3). (D, top) The expressions of ATF3, GDF15, NDRG1, KAI-1, and β-actin of ATF3-knockdown (HT_shATF3) or mock-knockdown (HT_shCOL) HT1376 cells were determined by immunoblot assays. (D, bottom) Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin relative to the mock-control group (±SE; n = 3). (E) The mRNA ratio of ATF3, GDF15, NDRG1, and KAI-1 between HT_shATF3 and HT_shCOL cells was determined by RT-qPCR. (F) The reporter activities of GDF15, NDRG1, and KAI-1 reporter vectors after transient overexpression of various dosages of the ATF3 expression vector, as indicated, were determined by reporter assays ( n = 6). (G) Cell growth rate following ATF3 knockdown was determined by Ki67 proliferation assays ( n = 3). ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Gene Expression, Western Blot, Produced, Control, Knockdown, Quantitative RT-PCR, Over Expression, Expressing, Plasmid Preparation

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of ATF3 on cell invasion and epithelial-to-mesenchymal transition. The invasion ability of (A) T24-DNA, T24-ATF3, (B) HT_shCOL, and HT_shATF3 cells was determined by Matrigel invasion assays. The quantitative analysis data were expressed as average cell counts/9 fields ± SE. (C) The expressions of N-cadherin, E-cadherin, Snail, Slug, and β-actin in HT_shCOL and HT_shATF3 cells were determined by immunoblot assays. (D) Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin relative to the mock-control (HT_shCOL) group (±SE; n = 3). (E) The F-actin staining with Texas Red X-Phalloidin and the fluorescence were recorded using a confocal microscope. (F) The intensities were measured along the line from the peripheral to the central area of the cells, and the quantitative analysis (G) of the F-actin fluorescence intensity of HT_shCOL and HT_shATF3 cells (±SE, n = 4). ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Western Blot, Produced, Control, Staining, Fluorescence, Microscopy

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of ATF3 on tumor growth of bladder cancer cells in xenograft model. (A) HT_shCOL and HT_shATF3 cells were injected subcutaneously in the dorsal area of the four-week-old male athymic nude mice (n = 8). Tumors derived from both cells were recorded after the mice were sacrificed. The tumor growth rates (B) and animal body weights (C) were measured within 24 days. (D) The tumor weights were recorded immediately after the sacrifice (±SE; n = 8). (E) The protein levels of ATF3, GDF15, NDRG1, KAI-1, and β-actin of the tumors derived from the HT_shCOL and HT_shATF3 cells were determined by immunoblot assays. (F) The quantitative analysis was presented as the relative density of the target proteins/β-actin (±SE; n = 5). ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Injection, Derivative Assay, Western Blot

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of metformin on the expressions of ATF3 and GDF15 in the bladder cancer cells. The expressions of ATF3, GDF15, NDRG1, and β-actin in the T24 cells after being treated with or without 4 mM of metformin at normal glucose conditions (5 mM) were determined by immunoblot assays (A) and quantitative analysis (B). The expressions of ATF3 and GDF15 in the HT1376 cells after being treated with 5 mM or 30 mM glucose and with/without 4 mM of metformin, as indicated. were determined by immunoblot assays (C) and quantitative analysis (D). (E) Gene expressions of ATF3 and GDF15 in HT1376 cells after being treated with/without various dosages of metformin, as indicated, were determined by RT-qPCR. Protein expressions of ATF3 and GDF15 in HT1376 cells after being treated with/without metformin or SB431542, as indicated, were determined by immunoblot assays (F) and quantitative analysis (G). (H) Protein expressions of ATF3 and GDF15 in HT_shCOL and HT_shATF3 cells after being treated with/without metformin were determined by immunoblot assays (left) and quantitative analysis (right). Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin (±SE; n = 3) relative to the vehicle-treated group. ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Western Blot, Quantitative RT-PCR, Produced

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Co-modulation between ATF3 and GDF15 in the bladder cancer cells. The expressions of ATF3, GDF15, and β-actin in (A) T24-DNA, T24-GDF15, (B) HT_shCOL, and HT_shGDF15 cells were determined by immunoblot assays. Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin (±SE; n = 3) relative to the vehicle-treated group. The ratio of gene expressions of ATF3 and GDF15 in (C) T24-DNA, T24-GDF15, (D) HT_shCOL, HT_shGDF15, (E) T24-DNA, T24-ATF3, (F) HT_shCOL, and HT_shATF3 cells were determined by RT-qPCR. Data from quantitative analysis were expressed as the expressions of the target genes/β-actin relative to the mock-control group (±SE; n = 3). ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Western Blot, Produced, Quantitative RT-PCR, Control