Journal: bioRxiv

Article Title: Differential antitumor activity of compounds targeting the ubiquitin-proteasome machinery in gastrointestinal stromal tumor (GIST) cells

doi: 10.1101/791426

Figure Lengend Snippet: (A) Relative tumor volume of UZLX-GIST1 xenografts over the course of a 21-day treatment with vehicle, delanzomib or imatinib. Measurements represent the average of at least four tumors per group. (B) Histopathologic response of imatinib-sensitive UZLX-GIST1 xenografts to treatment with delanzomib in comparison with placebo-treated and imatinib-treated controls. Hematoxylin and eosin (H&E) as well as immunohistochemical staining for Ki-67, phosphorylated histone H3 S10 or cleaved PARP (10X magnification). Histopathologic response grade was determined according to Agaram et al. . Data shown in graph represent at least six tumors per group. Quantification of proliferation index (% Ki-67 positive cells), mitotic cells (pH3-positive cells per 10 high-power fields, HPF) and percentage of cleaved PARP-positive cells (brown staining, respectively) represents the average of at least six tumors per group. It is of note, that only intact, individual cells were included in the counts. Hence, the central debris area in the cleaved PARP stain (although massively positive) is not reflected in the accompanying graph. Columns, mean + SE; *, p≤0.05 in comparison to control; **, p≤0.01 in comparison to control; ***, p≤0.001 in comparison to control (Student’s t-test, 2-tailed). (C) Immunoblot analysis of UZLX-GIST1 xenografts treated with delanzomib (24 h) in comparison with placebo and imatinib-treated positive control. Each lane represents one xenograft (C, vehicle control; DLZ, delanzomib; IM, imatinib). Grouped immunoblot images are either cropped from different parts of the same gel or from a separate gel run with another aliquot of the same protein lysate. (D) Histopathologic response of imatinib-resistant GIST430 xenografts to treatment with delanzomib in comparison with placebo. (E) Immunohistochemical staining for phosphorylated histone H3 S10 in GIST430 xenografts treated with delanzomib or placebo control.

Article Snippet: Primary antibodies used for immunoblotting, immunofluorescence and immunohistochemistry were actin (Sigma), cleaved caspase 3 (Cell Signaling), CREB binding protein (CBP; Santa Cruz), cyclin A (Novocastra), pH2AX S139 (Millipore), H2AX (Bethyl), phospho-histone H3 S10, phospho-KIT Y719 (both Cell Signaling), Ki-67 (Thermo Scientific Neomarkers), KIT (Dako/Agilent), p27 Kip1 (Fisher/BD Biosciences Pharmingen), PARP (Invitrogen), cleaved PARP (Abcam) and mono-ubiquitin (BD Pharmingen).

Techniques: Comparison, Immunohistochemical staining, Staining, Control, Western Blot, Positive Control

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