Journal: European journal of pharmacology

Article Title: Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence.

doi: 10.1016/j.ejphar.2024.176397

Figure Lengend Snippet: Fig. 3. TZ reduces AAA formation by attenuating PEG3 signaling. (A) Heatmap depicting differentially expressed genes between control and 10 nM TZ-treated MOVAS cells. (B) Representative images of IHC staining for PEG3 in human AAA samples (scale bar, 50 μm) and quantification of IHC results (n = 3). Black arrows indicate PEG3-positive cells. *p < 0.05 compared with the control group. (C) MOVAS cells were treated by 1 μM Ang II with or without TZ for 48 h. Representative western blots and quantification of PEG3 and MMP2 protein expressions in MOVAS (n = 3–6). *p < 0.05 compared with ctrl group, #p < 0.05 between Ang-TZ and Ang II group. (D) (Upper) Representative IHC staining images of PEG3 in Apoe−/−mice suprarenal aortas from each group (scale bar, 50 μm) and quantification of IHC results (n = 5–9). *p < 0.05 compared with the sham-water group, #p < 0.05 compared with the Ang II-water group. (Lower) Representative IHC staining images of PEG3 in C57BL/6J mice infrarenal aortas from each group (scale bar, 50 μm) and quantification of IHC results (n = 5–12). *p < 0.05 compared with the sham-water group, #p < 0.05 compared with the CaCl2-water group. Black arrows indicate PEG3-positive cells. (E) (Left) Representative Immunofluorescent staining images of PEG3 (Green), α-SMA (Red) and DAPI (Blue) in Apoe−/−mice suprarenal aortas from sham-water group, Ang II-water group elastin unbroken area, Ang II- water group elastin broken area and Ang II-TZ (100 μg//kg) group (scale bar, 50 μm). (Right) Representative Immunofluorescent staining images of PEG3 (Green), α-SMA (Red) and DAPI (Blue) in C57BL/6J mice infrarenal aortas from the indicated groups (scale bar, 50 μm). IHC, Immunohistochemistry. α-SMA, α-smooth muscle actin.

Article Snippet: Primary antibodies used included: PEG3 (AF9152, 1:150, Affinity, China), α-SMA (BM0002, 1:200, BOSTER, Wuhan, China), p21 (sc-6246, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (10375-2-AP, 1:300, Proteintech, Chicago, IL, USA), MMP2 (10373-2-AP, 1:400, Proteintech), and F4/80 (#70076, 1:200, CST, Boston, MA, USA).

Techniques: Control, Immunohistochemistry, Western Blot, Staining

Journal: European journal of pharmacology

Article Title: Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence.

doi: 10.1016/j.ejphar.2024.176397

Figure Lengend Snippet: Fig. 6. Silencing Peg3 alleviates Ang II-induced cell senescence and apoptosis in MOVAS. MOVAS cells were exposed to 1 μM Ang II with or without Peg3 knockdown for 48 h. (A) Representative western blots and (B) quantification of PEG3, MMP2, p53, cleaved caspase-3 p21, p16 and p-H2AX protein expressions (n = 3). (C) Relative mRNA levels of Peg3 analyzed by RT-qPCR (n = 6). (D) Relative mRNA levels of Mmp2, Mmp9, p53, p21 and p16 analyzed by RT-qPCR (n = 6). (E) (Left) Representative flow-cytometric plots of Annexin V-APC/7-AAD staining. (Right) Quantification of early apoptosis (Annexin V(+)/7-AAD(−)) and total apoptosis (Annexin V(+)) percentages (n = 6). (F) (Left) Representative images of SA-β-gal staining in MOVAS (scale bar, 50 μm) and (Right) quantification of SA- β-gal positive cells (n = 3). (E) Relative mRNA levels of SASP factors including Il-6, Il-1β, Ccl2, Ccl7, Cxcl1, Cxcl10 and Cxcl12 analyzed by RT-qPCR (n = 3). *p < 0.05 (si-Peg3 group vs NC group) or (si-Peg3+Ang II group vs NC + Ang II group), #p < 0.05 (NC + Ang II group vs NC group) or (si-Peg3+Ang II group vs si-Peg3 group).

Article Snippet: Primary antibodies used included: PEG3 (AF9152, 1:150, Affinity, China), α-SMA (BM0002, 1:200, BOSTER, Wuhan, China), p21 (sc-6246, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (10375-2-AP, 1:300, Proteintech, Chicago, IL, USA), MMP2 (10373-2-AP, 1:400, Proteintech), and F4/80 (#70076, 1:200, CST, Boston, MA, USA).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Staining

Journal: European journal of pharmacology

Article Title: Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence.

doi: 10.1016/j.ejphar.2024.176397

Figure Lengend Snippet: Fig. 7. Schematic illustration shows the mechanisms of TZ inhibition on AAA formation. Stress induces the expression of Peg3 in VSMCs, triggering VSMCs senescence, apoptosis, and ECM degradation, eventually leading to AAA formation Treatment with low-dose TZ reduces Peg3 expression, reversing these effects. The picture was drawn by Figdraw. VSMCs, vascular smooth muscle cells. SASP, senescence-associated secretory phenotype. ECM, extracellular matrix.

Article Snippet: Primary antibodies used included: PEG3 (AF9152, 1:150, Affinity, China), α-SMA (BM0002, 1:200, BOSTER, Wuhan, China), p21 (sc-6246, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (10375-2-AP, 1:300, Proteintech, Chicago, IL, USA), MMP2 (10373-2-AP, 1:400, Proteintech), and F4/80 (#70076, 1:200, CST, Boston, MA, USA).

Techniques: Inhibition, Expressing

Journal: eLife

Article Title: PIMT is a novel and potent suppressor of endothelial activation

doi: 10.7554/elife.85754

Figure Lengend Snippet: Figure 3. PIMT functionally interacts with TRAF6. (A) EA.hy926 cells were transfected with NF-κB luciferase reporter together with indicated plasmids. Cells were harvested 48 hr later to determine luciferase activity (n=4), ***p<0.001, and two-way ANOVA coupled with Tukey’s post hoc test. Expression of transfected proteins was detected by western blot. (B) HEK-293T cells were transfected with a combination of indicated plasmids. 48 hr after transfection, immunoprecipitation was performed using anti-Flag antibody, followed by western blot to detect protein interaction. (C) HEK-293T cells were transfected with Flag-TRAF6 and Myc-PIMT expression vectors. 48 hr after transfection, immunoprecipitation was performed using anti-Flag antibody, followed by western blot to detect the interaction of PIMT with TRAF6. (D) HEK-293T cells were transfected with HA-TRAF6 and Flag-PIMT vectors. 48 hr after transfection, immunoprecipitation was performed using anti-HA antibody, followed by western blot to detect protein interaction. (E) Human umbilical vein endothelial cells (HUVECs) were transduced with lentivirus expressing the control shRNA (sh-Ctrl) or TRAF6 shRNA (sh-TRAF6) for 72 hr, followed by stimulation with either vehicle or LPS (1 μg/ml) for 30 min. Cell lysates were then collected for immunoprecipitation using anti-PIMT

Article Snippet: AntiGAPDH (10494–1- AP), anti- alpha Tubulin (11224–1- AP), and anti- TRAF6 (66498–1- Ig) antibodies were acquired from Proteintech Group.

Techniques: Transfection, Luciferase, Activity Assay, Expressing, Western Blot, Immunoprecipitation, Transduction, Control, shRNA

Journal: eLife

Article Title: PIMT is a novel and potent suppressor of endothelial activation

doi: 10.7554/elife.85754

Figure Lengend Snippet: Figure 4. PIMT suppresses TRAF6 function through methylation of TRAF6 at N350. (A) HEK-293T cells were transfected with TRAF6 and PIMT constructs. Immunoprecipitation was performed 48 hr after transfection using anti-Flag antibody, followed by western blot using anti-HA antibody to detect TRAF6 oligomerization. (B) HEK- 293T cells were transfected with HA-ubiquitin (HA-Ub) and Flag-TRAF6 in the presence of Myc-tagged wild-type

Article Snippet: AntiGAPDH (10494–1- AP), anti- alpha Tubulin (11224–1- AP), and anti- TRAF6 (66498–1- Ig) antibodies were acquired from Proteintech Group.

Techniques: Methylation, Transfection, Construct, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: ZNF593 is overexpressed in breast cancer and is associated with advanced clinicopathological features (A) A pan-cancer analysis of ZNF593 mRNA expression level in 34 cancers. (B) Differential gene analysis in breast cancer according to the cancer genome atlas (TCGA) database (TCGA:BRCA). (C and D) Unpair comparison and pair comparison of ZNF593 expression in normal mammary tissues and BC. (E) The ZNF593’s PCR assay of five pairs of cancer and adjacent normal tissues from breast cancer. (F) IHC staining images of ZNF593 in breast cancer tissue and normal mammary tissue from the HPA database (HPA: https://www.proteinatlas.org ). (G) Expression of ZNF593 in BRCA based on nodal metastasis status. (H) Expression of ZNF593 in BRCA based on breast cancer subclasses. (I) Expression of ZNF593 based on TNBC (IHC) status. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001 by two-tailed Student’s t test or two-way ANOVA test. Data are represented as mean ± SD.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Expressing, Comparison, Immunohistochemistry, Two Tailed Test

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: Higher expression of ZNF593 is correlated with poor survival in breast cancer patients (A and B) Kaplan-Meier plots of ZNF593 in breast cancer according to overall survival (OS) and relapse-free survival (RFS) (KM plotter: https://kmplot.com/analysis/ ). Log rank test. (C and D) OS and RFS of breast cancer patients analyzed by the GSE1456 dataset (GEO: GSE1456 ). Log rank test. (E) IHC staining analysis of ZNF593 expression in 66 breast cancer patients. Representative images are shown. Scale bar: 100 μm. The scale length of the small image in the bottom right corner of each picture is 50 μm. (F and G) Higher expression level of ZNF593 is associated with poor OS and disease-free survival (DFS). Log rank test.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Expressing, Immunohistochemistry

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: ZNF593 deficiency inhibits cell proliferation and migration in breast cancer (A and B) ZNF593 expression was confirmed via RT-qPCR analysis in MDA-MB-231 and SUM159PT cells transfected with two distinct siRNAs targeting ZNF593. (C and D) Western blot verified that both of the above two distinct siRNAs could knock down ZNF593 at the protein level. (E and F) Knockdown of ZNF593 suppresses cell proliferation in MDA-MB-231 and SUM159PT cells, as assessed by cell counting kit-8 assays. (G) Knockdown of ZNF593 suppresses migration ability of MDA-MB-231 and SUM159PT cells by migration assays. Scale bar: 100 μm. (H and I) Corresponding quantitative results of migration ability in MDA-MB-231 and SUM159PT cells are shown. ∗∗ p < 0.01; ∗∗∗ p < 0.001 by two-tailed Student’s t test or two-way ANOVA test. Data are represented as mean ± SD.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Migration, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Knockdown, Cell Counting, Two Tailed Test

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: Co-expression analysis, enrichment analysis, and protein-protein interaction analysis of ZNF593 (A and B) The top 50 genes positively and negatively correlated with ZNF593 expression by Linkedomics (Linkedomics: http://www.linkedomics.org/login.php ). (C) GO and KEGG enrichment analyses of ZNF593 and its related 100 genes. (D) PPI analysis on ZNF593 in breast cancer.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Expressing

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: ZNF593 is strongly connected with the DNA damage repair (A–D) Enrichment plot of ZNF593 about DNA damage repair by GSEA according to TCGA database. (E and F) ssGSEA of ZNF593 in TCGA and FUSCC-TNBC database about DNA damage repair. (G and H) Western blot experiments in MDA-MB-231 and SUM159PT cells confirmed that ZNF593 is involved in DNA damage repair. (I and J) ZNF593 affects DNA damage repair by affecting RAD50 expression. (K and L) Drug sensitivity assays of MDA-MB-231 cells before and after ZNF593 knockdown with cisplatin and carboplatin. (M and N) Drug sensitivity assays of SUM159PT cells before and after ZNF593 knockdown with cisplatin and carboplatin. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 by two-tailed Student’s t test or two-way ANOVA test. Data are represented as mean ± SD.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Western Blot, Expressing, Knockdown, Two Tailed Test

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: ZNF593 regulates cell-cycle progression in breast cancer cell (A–D) Enrichment plot of ZNF593 about cell-cycle progression by GSEA according to TCGA database. (E–G) GSEA of ZNF593 in GSE1456 and FUSCC-TNBC database about cell-cycle progression. (H) Knockdown of ZNF593 results in cell-cycle arrest at the G1 phase in MDA-MB-231 and SUM159PT cells, as demonstrated by flow cytometry analysis using propidium iodide (PI) staining. The percentage of cells in the G1/S/G2 phase are shown. (I and J) Corresponding quantitative results of flow cytometry analysis in MDA-MB-231 and SUM159PT cells are shown. (K and L) Knockdown of ZNF593 represses the mRNA expression of CCND1, CCNE1, and CCNA2 in MDA-MB-231 and SUM159PT cells by RT-qPCR analysis. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 by two-tailed Student’s t test or two-way ANOVA test. Data are represented as mean ± SD.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Knockdown, Flow Cytometry, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet: ZNF593 inhibits the formation of tumor immune environment (A) ESTIMATE score analysis in breast cancer according to ZNF593 expression level. (B and C) ssGSEA of ZNF593 in TCGA and FUSCC-TNBC database about immune environment. (D) Percentage of 22 types of immune cells in breast cancer patients. (E, H, K, and N) Correlation between many immune cell markers and ZNF593 in breast cancer tissues. (F and G, I and J, L and M, and O and P) RT-qPCR was used to verify the relationship between the immune markers and ZNF593. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 by two-tailed Student’s t test or two-way ANOVA test. Data are represented as mean ± SD.

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

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

Journal: iScience

Article Title: Zinc finger protein 593 promotes breast cancer development by ensuring DNA damage repair and cell-cycle progression

doi: 10.1016/j.isci.2024.111513

Figure Lengend Snippet:

Article Snippet: ZNF593 Polyclonal antibody , Proteintech , Cat# 19426-1-AP; RRID: AB_10642948.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Cell Counting, Gene Expression, Preserving, Software

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: Infection of Nsp15 WT and mutant SARS-CoV-2 in cell culture. ( a ) Schematic diagram of the recombinant SARS-CoV-2 genome with Nsp15 H234A and N277A mutations. ( b ) Replication kinetics of Nsp15 WT , Nsp15 H234A and Nsp15 N277A rSARS-CoV-2 in Vero E6 cells (m.o.i. of 0.001). ( c ) Cell lysates from mock- and virus-infected Vero E6 cells (m.o.i. of 0.001) after 48 h of infection were incubated with anti-Nsp15 antibody or isotype control. Immunoblot analysis was applied for the immunoprecipitated Nsp15 proteins. ( d ) Western blot for ACE2 and STAT1 in A549-ACE2 cells with or without STAT1 knockout. ( e, f ) Replication kinetics of Nsp15 WT , Nsp15 H234A and Nsp15 N277A rSARS-CoV-2 in A549-ACE2 and A549-ACE2/STAT1 knockout cells (m.o.i. of 1). ( g ) The AUC was calculated from each viral growth curve and plotted as a bar graph. Data are mean±sd ( n =3) and analysed by two-way ANOVA with Šídák multiple comparison test. ( h ) A549-ACE2 cells were uninfected or infected by Nsp15 WT , Nsp15 H234A and Nsp15 N277A rSARS-CoV-2 at m.o.i. of 1 for 8 and 24 h. Total RNA was collected for evaluating the host responses by RT-qPCR. Relative gene expression was normalized by 18S rRNA and presented relative to mock infection. Data are mean±sd ( n =3) and analysed by two-way ANOVA with Dunnett’s multiple comparison test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001; **** P ≤0.0001; ns, not significant.

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Infection, Mutagenesis, Cell Culture, Recombinant, Virus, Incubation, Control, Western Blot, Immunoprecipitation, Knock-Out, Comparison, Quantitative RT-PCR, Gene Expression

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: Viral pathogenesis of Nsp15 EndoU-inactive SARS-CoV-2 in hamsters. Syrian hamsters were intranasally inoculated with 10,000 p.f.u. of Nsp15 WT or Nsp15 H234A rSARS-CoV-2, or equivalent volume of PBS. ( a ) Body weight change (%) following mock, Nsp15 WT and Nsp15 H234A virus infection ( n =4). ( b ) Viral titre in the nasal turbinates, lungs, olfactory bulbs and brains at 5 dpi, measured by plaque assay. Data are mean± sem ( n =4) and analysed by unpaired t-test. * P ≤0.05; ** P ≤0.01. The dotted line represents the limit of detection. ( c ) Host responses in upper lungs (cranial) at 5 dpi assessed by RT-qPCR. Data are mean± sem ( n =4, except for mock/PBS where n =3). Exact P -values are shown and calculated by a parametric unpaired t-test. ( d ) Lung pathology assessment at 5 dpi performed by H&E staining. Scale bar=500 µm.

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Virus, Infection, Olfactory, Plaque Assay, Quantitative RT-PCR, Staining

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: The global transcriptional signatures from Nsp15 WT and mutant SARS-CoV-2-infected A549-ACE2 cells. A549-ACE2 cells with mock, Nsp15 WT , Nsp15 H234A and Nsp15 N277A rSARS-CoV-2 infection for 8 and 24 h (m.o.i. of 5). Total RNA with poly(A) enrichment followed by RNA-seq analysis. ( a ) Schematic of bulk RNA-seq experimental design ( n =3 per group). ( b ) PCA of total normalized transcript abundance from mock, Nsp15 WT and mutant rSARS-CoV-2 infection. Sparse PCA depicts the global transcriptome of an individual sample. ( c ) Venn diagram for unique and shared differentially expressed genes ( P adj<0.05 and |log2FC|>1) in cells infected with Nsp15 H234A and Nsp15 N277A mutants compared to Nsp15 WT virus. ( d ) Volcano plots showing GSEA results generated using MSigDB Hallmark pathways. ( e ) Cluster heatmap of GSVA scores generated using representative innate immune and metabolic signatures from MSigDB Gene Ontology signatures. ( f ) Expression heatmap of representative innate immune and metabolic genes across mock, WT and mutant rSARS-CoV-2 infection.

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Mutagenesis, Infection, RNA Sequencing, Virus, Generated, Expressing

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: Decrease in cGAS and STING during SARS-CoV-2 infection. ( a ) Endogenous cGAS and STING mRNA and protein levels in A549-ACE2 cells uninfected or infected with Nsp15 WT and Nsp15 H234A rSARS-CoV-2 at m.o.i. of 5 for 24 h. RT-qPCR results are presented relative to the expression of 18S rRNA. Data are mean± sd ( n =3) and analysed by one-way ANOVA with Tukey’s multiple comparison test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. ( b ) Endogenous cGAS and STING protein levels in mock, Nsp15 WT and Nsp15 H234A rSARS-CoV-2 (m.o.i. of 5) infected A549-ACE2/STAT1 knockout cells at 24 hpi.

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Infection, Quantitative RT-PCR, Expressing, Comparison, Knock-Out

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: Inhibition of host cGAS–STING pathway by Nsp15. ( a ) Western blot for ACE2 and STING in A549-ACE2 cells with or without STING knockout. ( b ) Replication kinetics of Nsp15 WT and Nsp15 H234A rSARS-CoV-2 (m.o.i. of 1) in A549-ACE2 and A549-ACE2/STING knockout cells. Viral supernatants collected at individual time points for titration by plaque assay. Data are mean± sd ( n =3) and analysed by two-way ANOVA with Tukey’s multiple comparison test. ( c, d ) HEK293T cells were co-transfected with IFN- β promoter reporter plasmid ( c ) or NF- κ B responsive element reporter plasmid ( d ), Renilla control plasmid plus the indicated plasmids containing empty vector, cGAS/STING, mCherry and WT and H234A Nsp15 for 48 h. Relative luciferase activity was performed by use of Dual-Glo Luciferase System. Data are mean± sd ( n =3) and analysed by two-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Inhibition, Western Blot, Knock-Out, Titration, Plaque Assay, Comparison, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: Role of Nsp15 EndoU activity in cGAS and STING downregulation. ( a, b ) HEK293T cells were transfected with cGAS or STING plasmid along with WT and mutant SARS-CoV-2 Nsp15 plasmids for 48 h. Protein levels of cGAS ( a ) and STING ( b ) were measured by Western blot. ( c, d ) HEK293T cells were transfected with cGAS and STING plasmids along with SARS-CoV-2 Nsp5-FLAG or Nsp15-FLAG plasmids. After 24 h, total cell lysates were collected and incubated with anti-DYKDDDDK magnetic agarose to pull down the FLAG-tagged proteins and interacting RNAs. ( c ) Western blot assay of the immunoprecipitated and input Nsp5 and Nsp15 proteins. ( d ) The Nsp5 and Nsp15-binding cGAS, STING and 18S RNA were quantified using RT-qPCR. The target RNA levels were presented as the fold change in the Nsp15 group relative to the Nsp5 control. Data are mean± sd ( n =3) and analysed by unpaired t-test. ** P ≤0.01; ns , not significant. ( e ) HEK293T cells transfected with mCherry, WT or H234A Nsp15 plasmids for 48 h were treated with actinomycin D (5 µg ml −1 ). Samples were collected at the designated time points post-treatment to monitor the stability of cGAS and STING mRNAs using RT-qPCR. Data are mean± sd ( n =3) and shown as relative mRNA expression compared to the starting point (0 h, without ActD).

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Incubation, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Control, Expressing

Journal: The Journal of General Virology

Article Title: SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways, including cGAS–STING

doi: 10.1099/jgv.0.002233

Figure Lengend Snippet: Divergent activity in downregulating cGAS and STING among human CoV Nsp15. ( a, b ) HEK293T cells were transfected with cGAS or STING plasmid plus mCherry or HCoV Nsp15 plasmids for 48 h. Western blot performed to assess the expression of indicated proteins. ( c ) Protein analysis of the HEK293T cells transfected with cGAS or STING plasmid along with mCherry, SARS-CoV-2 and HCoV-229E Nsp15 (WT and corresponding EndoU mutant) plasmids for 48 h.

Article Snippet: Cells (4 mg of lysates) lysed by RIPA lysis buffer (Pierce) containing protease inhibitor cocktail (Roche) were immunoprecipitated with pre-incubated mixture of 50 μl of protein A/G magnetic beads (Pierce) and 10 μg of anti-SARS-CoV-2 Nsp15 antibody (16820-1-AP, Proteintech) or rabbit IgG (12–370, EMD Millipore) at 4 °C overnight.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Mutagenesis

Journal: Frontiers in Molecular Neuroscience

Article Title: Transcription factor ZNF22 regulates blood-tumor barrier permeability by interacting with HDAC3 protein

doi: 10.3389/fnmol.2022.1027942

Figure Lengend Snippet: Analysis using bioinformatics. (A) GO analysis of HDAC family and ZNF22. (B) GDC database-based study of the relative expression of ZNF22 in gliomas. (C) GDC database-based study of the relative expression of HDAC3 in gliomas. (D) Survival curves of glioma patients with high HDAC3 expression based on the GDC database.

Article Snippet: The sealant was rinsed with TTBS, and the primary antibody diluent was used to dilute the primary antibody: ZNF22 1:1000 and HDAC3 1:1000 (proteintech, Beijing, China), ZO-1 1:300 and Occludin 1:200 and Claudin-5 1:500 (Thermo Scientific, Beijing, China) in certain proportions, sealed in film and left overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Transcription factor ZNF22 regulates blood-tumor barrier permeability by interacting with HDAC3 protein

doi: 10.3389/fnmol.2022.1027942

Figure Lengend Snippet: Endogenous expression of ZNF22 was detected in GECs of BTB and was involved in the regulation of BTB permeability in vitro . (A) qRT-PCR was used to identify the relative expression of ZNF22 in ECs and GECs. As an endogenous control, GAPDH protein levels were measured. Data represent mean ± SD ( n = 3, each). *** p < 0.001 vs. ECs group. (B) Test for the expression of ZNF22 protein using immunoblotting. As an endogenous control, GAPDH protein levels were measured. Data represent mean ± SD ( n = 3, each). ** p < 0.01 vs. ECs group. (C) TEER values of GECs were expressed as Ωcm 2 . Data were expressed as mean ± SD ( n = 3, each). *** p < 0.001 vs. ZNF22(–) NC group. (D) pmol/cm 2 /h was used to compute the HRP flow. Data were expressed as mean ± SD ( n = 3, each). *** p < 0.001 vs. ZNF22(–) NC group. (E) TJ-associated proteins ZO-1, Occludin, and Claudin-5 in GECs were examined by protein blotting. As an endogenous control, GAPDH protein levels were measured. Data were expressed as. * p < 0.05 and ** p < 0.01 vs. ZNF22(–) NC group. (F) ZO-1, Occludin, and Claudin-5 immunofluorescent localization in GECs. ZO-1, Occludin, and Claudin-5 all were colored red using fluorescent secondary antibodies, and DAPI was used to mark the nuclei. Three separate experiments were shown by the photographs. Scale bar = 20 μm.

Article Snippet: The sealant was rinsed with TTBS, and the primary antibody diluent was used to dilute the primary antibody: ZNF22 1:1000 and HDAC3 1:1000 (proteintech, Beijing, China), ZO-1 1:300 and Occludin 1:200 and Claudin-5 1:500 (Thermo Scientific, Beijing, China) in certain proportions, sealed in film and left overnight at 4°C.

Techniques: Expressing, Permeability, In Vitro, Quantitative RT-PCR, Control, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Transcription factor ZNF22 regulates blood-tumor barrier permeability by interacting with HDAC3 protein

doi: 10.3389/fnmol.2022.1027942

Figure Lengend Snippet: In the GECs of the BTB, ZNF22 binds to the promoters of TJ-associated proteins. (A) The human ZO-1 promoter regions are shown schematically at 3,000 bp upstream of the transcription start site (marked as TSS, + 1). Using their unique primers, ChIP PCR results for binding sites and upstream areas not anticipated to connect with ZNF22 were amplified by PCR. The Images served as examples of separate ChIP studies. (B) Results of three independent ChIP-qPCRs to confirm the presence of ZNF22 binding to specific regions of the promoter of ZO-1. *** p < 0.001 vs. IgG group. (C) The human Occludin promoter regions are shown schematically in 3,000 bp increments upstream of the transcription start site (TSS, denoted as + 1). Using specified primers, ChIP PCR results for binding locations and upstream areas not anticipated to connect with ZNF22 were amplified. The images were taken from independent ChIP tests. (D) Results of three independent ChIP-qPCRs to confirm the presence of ZNF22 binding to specific regions of the promoter of Occludin. *** p < 0.001 vs. IgG group. (E) The human Claudin-5 promoter regions are shown schematically in 3,000 bp increments upstream of the transcription start site (TSS, denoted as + 1). Using specified primers, ChIP PCR results for binding locations and upstream areas not anticipated to connect with ZNF22 were amplified. The images were taken from independent ChIP tests. (F) Results of three independent ChIP-qPCRs to confirm the presence of ZNF22 binding to specific regions of the promoter of Claudin-5. * p < 0.05 vs. IgG group. (G) ZNF22 and ZO-1 binding sites in GECs were identified using dual luciferase reporter assays. Data represent mean ± SD (n = 3, each). * p < 0.05 vs. ZNF22(+) NC group. ZNF22(+) NC group. (H) The binding sites of ZNF22 and Occludin in GECs were determined using dual luciferase reporter assays. Data represent mean ± SD ( n = 3, each). * p < 0.05 vs. ZNF22(+) NC group. (I) ZNF22 and Claudin-5 binding sites in GECs were determined using dual luciferase reporter assays. Data represent mean ± SD ( n = 3, each). * p < 0.05 vs.

Article Snippet: The sealant was rinsed with TTBS, and the primary antibody diluent was used to dilute the primary antibody: ZNF22 1:1000 and HDAC3 1:1000 (proteintech, Beijing, China), ZO-1 1:300 and Occludin 1:200 and Claudin-5 1:500 (Thermo Scientific, Beijing, China) in certain proportions, sealed in film and left overnight at 4°C.

Techniques: Binding Assay, Amplification, Luciferase

Journal: Frontiers in Molecular Neuroscience

Article Title: Transcription factor ZNF22 regulates blood-tumor barrier permeability by interacting with HDAC3 protein

doi: 10.3389/fnmol.2022.1027942

Figure Lengend Snippet: ZNF22 interacted with HDAC3 to block the expression of TJ-associated proteins, which led to activating BTB permeability. (A) Interaction between endogenous ZNF22 and HDAC3 was detected by immunoprecipitation in GECs. (B) Protein blot analysis of HDAC3 expression in GECs. As an endogenous control, GAPDH protein levels were measured. Data were expressed as mean ± SD ( n = 3, each). * p < 0.05 vs. ZNF22(–) NC group. (C) Protein blot analysis of ZNF22 expression in GECs. As an endogenous control, GAPDH protein levels were measured. Data represent mean ± SD ( n = 3, each). ** p < 0.01 vs. ZNF22(–) NC group. (D) In GECs, protein blot analysis revealed the expression of TJ-associated proteins ZO-1, Occludin, and Claudin-5. As an endogenous control, GAPDH protein levels were measured. The data is presented as mean ± SD ( n = 3, each). * p < 0.05 and ** p < 0.01 vs. HDAC3(–) NC + ZNF22(–) NC group. # p < 0.05 vs. HDAC3(–) group. & p < 0.05 vs. ZNF22(–) group. (E) ZO-1, Occludin, and Claudin-5 immunofluorescence localization in GECs. Fluorescent secondary antibodies were used to label ZO-1 (red), Occludin (red), and Claudin-5 (red), while DAPI was used to label nuclei. The photos were taken from three separate trials. Scale bar = 20 μm.

Article Snippet: The sealant was rinsed with TTBS, and the primary antibody diluent was used to dilute the primary antibody: ZNF22 1:1000 and HDAC3 1:1000 (proteintech, Beijing, China), ZO-1 1:300 and Occludin 1:200 and Claudin-5 1:500 (Thermo Scientific, Beijing, China) in certain proportions, sealed in film and left overnight at 4°C.

Techniques: Blocking Assay, Expressing, Permeability, Immunoprecipitation, Control, Immunofluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Transcription factor ZNF22 regulates blood-tumor barrier permeability by interacting with HDAC3 protein

doi: 10.3389/fnmol.2022.1027942

Figure Lengend Snippet: Schematic diagram of ZNF22 and HDAC3 to regulate BTB permeability.

Article Snippet: The sealant was rinsed with TTBS, and the primary antibody diluent was used to dilute the primary antibody: ZNF22 1:1000 and HDAC3 1:1000 (proteintech, Beijing, China), ZO-1 1:300 and Occludin 1:200 and Claudin-5 1:500 (Thermo Scientific, Beijing, China) in certain proportions, sealed in film and left overnight at 4°C.

Techniques: Permeability

Journal: Frontiers in Microbiology

Article Title: Intestinal Flora-Derived Kynurenic Acid Protects Against Intestinal Damage Caused by Candida albicans Infection via Activation of Aryl Hydrocarbon Receptor

doi: 10.3389/fmicb.2022.934786

Figure Lengend Snippet: KynA suppressed the MK2/p-MK2 signaling pathway by activating AHR. We investigated the regulation of TTP and expression changes of MK2 and P-MK2 induced by AHR activation at the cellular level. In Caco-2 cells, after treatment with KynA, expression of CYP1A1 increased significantly, as did expression of TTP also ( P < 0.05), while expression of MK2 decreased significantly ( P < 0.05). In the CA- Caco-2 cell model, the expression of proteins CYP1A1 and TTP decreased significantly, but the expression of p-MK2 increased ( P < 0.01). After the treatment of Caco-2 cells with KynA and the AHR agonist FICZ, the expression of CYP1A1 and TTP was significantly increased, but the expression of p-MK2 decreased significantly ( P < 0.01). Treatment with an AHR inhibitor (CH223191) led to a decrease in the expression of CYP1A1 and TTP, and an increase in the expression of p-MK2 ( P < 0.01). Statistical significance was evaluated using the Mann–Whitney U test. P -values < 0.05 (*) or < 0.01 (**) were considered statistically significant. CA, Candida albicans infection; KynA, Kynurenic acid; FICZ, 6-Formylindolo[3,2-b]carbazole. P -values < 0.001 (***).

Article Snippet: Proteintech, United States); anti-AHR antibody (1:1,000, 67785-1-Ig; Proteintech, United States); anti-ZO-1 antibody (1:1,000, 21773-1-AP, Proteintech, United States); anti-GAPDH antibody (1:1,000, 60004-1-Ig, Proteintech, United States); anti-MK2 antibody (1:1,000, 13949-1-AP, Proteintech, United States); anti-CYP1A1 antibody (1:1,000, 13241-1-AP, Proteintech, United States); anti-TTP antibody (1:1,000, 12737-1-AP; Proteintech, United States); anti-MLCK antibody (1:1,000, 21642-1-AP; Proteintech, United States); and anti-pMLC antibody (1:2,000, CST-3671; Cell Signaling Technology, United States).

Techniques: Expressing, Activation Assay, MANN-WHITNEY, Infection