Journal: Acta Pharmacologica Sinica

Article Title: CircSARS-CV2-N1368 from SARS-CoV-2 impairs endothelial cell function through the upregulation of ATF7 to activate TLR4/NF-κB/ROS signaling

doi: 10.1038/s41401-025-01516-8

Figure Lengend Snippet: a Detection of acetylcholine-induced endothelium-dependent vascular relaxation in rat coronary arteries with overexpression of circSARS-CV2-Ns (1-way ANOVA, n = 4 per group). b VEGFA expression and e-NOS activation (eNOS(S1177) phosphorylation) in rat coronary arteries by Western blot assay. c VEGFA expression, e-NOS and HH3 activation (phosphorylation of HH3) in circSARS-CV2-Ns-overexpressiong HCMECs by Western blot assay (1-way ANOVA, n = 3 per group). d Proliferation activity analysis of HCMECs by EdU assay (1-way ANOVA, n = 3 per group). Migration activity analysis of HCMECs by trans-well migration assay ( e ) and wound healing assay ( f ) (1-way ANOVA, n = 3 per group), respectively. g Microscopic images showing matrigel tube formation of HCMECs with overexpression of circSARS-CV2-Ns (1-way ANOVA, n = 3 per group). h Detection of ROS level by using DCFH-DA probe (1-way ANOVA, n = 3 per group). i Effect of exogenous overexpression of circSARS-CV2-Ns on angiogenesis in vivo by matrigel plug assay (1-way ANOVA, n = 5 per group). j , k Effects of exogenous overexpression of circSARS-CV2-Ns on size and beat of COs (2-way ANOVA, n = 5 per group). l Detection of eNOS and VEGFA mRNA expression by RT-qPCR assay (2-way ANOVA, n = 3 per group). The scale bar is 50 μm in ( d ), ( e ), ( h ), ( i ), the scale bar is 100 μm in ( g ), the scale bar is 200 μm in ( f ), ( j ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The membranes containing the target proteins were labeled with primary antibodies against ANP, β-MHC, α-SMA, p-e-NOS, e-NOS, VEGFA (Abcam), COL1A1, COL3A1, ATF7, TLR4, p-NF-κB p65, NF-κB p65, GAPDH (Proteintech), p-HH3, and HH3 (Cell Signaling Technology).

Techniques: Over Expression, Expressing, Activation Assay, Phospho-proteomics, Western Blot, Activity Assay, EdU Assay, Migration, Wound Healing Assay, In Vivo, Matrigel Assay, Quantitative RT-PCR

Journal: Acta Pharmacologica Sinica

Article Title: CircSARS-CV2-N1368 from SARS-CoV-2 impairs endothelial cell function through the upregulation of ATF7 to activate TLR4/NF-κB/ROS signaling

doi: 10.1038/s41401-025-01516-8

Figure Lengend Snippet: a Effect of miR-103a-3p on expression of VEGFA, e-NOS and HH3 activation in circSARS-CV2-N1368-overexpressiong HCMECs by Western blot assay. b MiR-103a-3p decreased ROS level in circSARS-CV2-N1368 overexpressing HCMECs. c Proliferation activity analysis of HCMECs by EdU assay. d , e Migration activity analysis of HCMECs by trans-well migration assay and wound healing assay, respectively. f Matrigel tube formation assay of circSARS-CV2-Ns-overexpressiong HCMECs with transfection of miR-103a-3p. Comparisons were made with 1-way ANOVA, n = 3 per group in ( b )–( f ). The scale bar is 50 μm in ( b ), (c ), ( e ), the scale bar is 100 μm in ( f ), the scale bar is 200 μm in ( d ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes containing the target proteins were labeled with primary antibodies against ANP, β-MHC, α-SMA, p-e-NOS, e-NOS, VEGFA (Abcam), COL1A1, COL3A1, ATF7, TLR4, p-NF-κB p65, NF-κB p65, GAPDH (Proteintech), p-HH3, and HH3 (Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Western Blot, Activity Assay, EdU Assay, Migration, Wound Healing Assay, Tube Formation Assay, Transfection

Journal: Acta Pharmacologica Sinica

Article Title: CircSARS-CV2-N1368 from SARS-CoV-2 impairs endothelial cell function through the upregulation of ATF7 to activate TLR4/NF-κB/ROS signaling

doi: 10.1038/s41401-025-01516-8

Figure Lengend Snippet: a Venn diagram showing the overlapped genes. b and c The expression of the concerned genes in HCMECs with transfection of miR-103a-3p by RT-qPCR assay. d Validation of the interaction between miR-103a-3p and Atf7 mRNA by RIP assay. e Determination of ATF7 and VEGFA expression, activations of e-NOS, HH3 and NF-κB p65 in HCMECs by Western blot assay. f MiR-103a-3p and si-Atf7 decreased ROS generation in circSARS-CV2-N1368 overexpressing HCMECs. g Proliferation activity analysis of HCMECs by EdU assay. h, i Migration activity of HCMECs by trans-well migration assay and wound healing assay, respectively. j Matrigel tube formation assay of circSARS-CV2-Ns-overexpressiong HCMECs with transfection of miR-103a-3p or si-Atf7. Comparisons were made with unpaired t test in ( b )–( d ), and comparisons were made with 1-way ANOVA in ( e )–( i ), n = 3 per group. The scale bar is 50 μm in ( f )–( h ), the scale bar is 100 μm in i , the scale bar is 200 μm in ( j ). ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes containing the target proteins were labeled with primary antibodies against ANP, β-MHC, α-SMA, p-e-NOS, e-NOS, VEGFA (Abcam), COL1A1, COL3A1, ATF7, TLR4, p-NF-κB p65, NF-κB p65, GAPDH (Proteintech), p-HH3, and HH3 (Cell Signaling Technology).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Activity Assay, EdU Assay, Migration, Wound Healing Assay, Tube Formation Assay

Journal: Acta Pharmacologica Sinica

Article Title: CircSARS-CV2-N1368 from SARS-CoV-2 impairs endothelial cell function through the upregulation of ATF7 to activate TLR4/NF-κB/ROS signaling

doi: 10.1038/s41401-025-01516-8

Figure Lengend Snippet: a Volcano plot showing the dysregulated genes in HCMECs by circSARS-CV2-N1368 and ATF7 siRNA, respectively. CircSARS-CV2-N1368-up-regulated genes and ATF7 siRNA-down-regulated genes in HCMECs were overlapped in Venn diagram ( b ) and categorized by KEGG enrichment analysis ( c ). d , e The expression of Toll-like receptor-related genes in HCMECs with overexpression of circSARS-CV2-N1368 or knock-down of ATF7 by RT-qPCR assay. f Transcriptional activation of TLR4 gene by dual luciferase assay. g Detection of ATF7, TLR4 and NF-κB p65 in circSARS-CV2-N1368 overexpressing HCMECs with immunofluorescence assay. h Expression of ATF7, TLR4 and NF-κB p65 activation in HCMECs by Western blot assay. i VEGFA expression, activations of eNOS, HH3 and NF-κB p65 in HCMECs by Western blot assay. j Detection of ROS level in HCMECs by using DCFH-DA probe. k Proliferation activity of HCMECs by EdU assay. l , m Migration activity of HCMECs by trans-well migration assay and wound healing assay, respectively. n Matrigel tube formation assay of circSARS-CV2-N1368-overexpressiong HCMECs with knock-down of ATF7, TLR4 and NF-κB p65, respectively. Comparisons were made with unpaired t test in ( d )–( f ), and comparisons were made with 2-way ANOVA in ( h ), ( i ), and 1-way ANOVA in ( j )–( n ), n = 3 per group. The scale bar is 50 μm in ( g ), ( j ), and ( l ), the scale bar is 100 μm in ( n ), the scale bar is 200 μm in ( m ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes containing the target proteins were labeled with primary antibodies against ANP, β-MHC, α-SMA, p-e-NOS, e-NOS, VEGFA (Abcam), COL1A1, COL3A1, ATF7, TLR4, p-NF-κB p65, NF-κB p65, GAPDH (Proteintech), p-HH3, and HH3 (Cell Signaling Technology).

Techniques: Expressing, Over Expression, Knockdown, Quantitative RT-PCR, Activation Assay, Luciferase, Immunofluorescence, Western Blot, Activity Assay, EdU Assay, Migration, Wound Healing Assay, Tube Formation Assay

Journal: Acta Pharmacologica Sinica

Article Title: CircSARS-CV2-N1368 from SARS-CoV-2 impairs endothelial cell function through the upregulation of ATF7 to activate TLR4/NF-κB/ROS signaling

doi: 10.1038/s41401-025-01516-8

Figure Lengend Snippet: a Detection of ROS level in circSARS-CV2-N1368 overexpressing HCMECs by using DCFH-DA probe. b Western blotting analysis of VEGFA expression, activations of e-NOS and HH3 in NAC-treated HCMECs with overexpression of circSARS-CV2-N1368. c Proliferation activity analysis of HCMECs by EdU assay. d , e Migration activity analysis of HCMECs was evaluated by trans-well migration assay and wound healing assay, respectively. f NAC rescued matrigel tube formation of HCMECs with overexpression of circSARS-CV2-N1368. Comparisons were made with 1-way ANOVA, n = 3 per group in ( a )–( f ), and 2-way ANOVA, n = 3 per group in ( b ), The scale bar is 50 μm in ( a ), ( c ), ( d ), the scale bar is 100 μm in ( f ), the scale bar is 200 μm in ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes containing the target proteins were labeled with primary antibodies against ANP, β-MHC, α-SMA, p-e-NOS, e-NOS, VEGFA (Abcam), COL1A1, COL3A1, ATF7, TLR4, p-NF-κB p65, NF-κB p65, GAPDH (Proteintech), p-HH3, and HH3 (Cell Signaling Technology).

Techniques: Western Blot, Expressing, Over Expression, Activity Assay, EdU Assay, Migration, Wound Healing Assay