Journal: EBioMedicine

Article Title: Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro.

doi: 10.1016/j.ebiom.2021.103712

Figure Lengend Snippet: Figure 3. SARS-CoV-2 induces N-glycosylation through NF-kB/STT3A axis. (a) Locus compare plot for the comparison between the STT3A eQTLs and the GWAS result, the COVID-19 A2 analysis for patients with severe respiratory symptoms via COVID-19 Host Genetics Initiative (COVID-19-HGI) project. The points are colored based on linkage disequilibrium (LD) bins. rs201008304, the indel variant in strong LD with the lead STT3A eQTL, is the top and most significant SNP in COVID-19 A2 analysis. (b) The eQTL plot for the association between STT3A variant types [A/A (n = 437), A/AT (n = 46), and AT/AT (n = 0)] expression and rs201008304 in cells-cultured fibroblasts was queried from the GTEx. (c) Forest plot shows the risk of rs201008304 in different COVID-19 phenotypes, originally from the summarized statistic results. (d) Representative data of immunohistochemistry staining of STT3A (red cells) and N protein in lung tissues of AAV-Ctrl and AAV-hACE2 mice infected by SARS-CoV2 at 5 dpi. Images were captured at 40x objective lens magnification. Scale bar, 100 mm. Three randomly selected STT3A/nuclei expression fields in lung tissues were exported from CaseViewer software (3DHistech) and quantified by the HistoQuant plugin in QuantCenter (3DHistech). Statistical method: t test, *p < 0.05, (n = 3). (e) Western blotting of S protein in Vero E6 cells after knockdown of STT3A or STT3B. Circle, g-spike; arrow, ng-spike. (f) ChIP analysis of enrichment of NF-kB in STT3A promoters after SARS-CoV-2 infection for 1 d. The enrichment levels were analyzed by RT-qPCR and shown as the per- centage of input. Statistical method: two-way ANOVA, Tukey post hoc tests, ***p < 0.001 (n = 3 with mean § SD shown).

Article Snippet: The deleted NF-kB binding motif construct had a deletion at bases 363CGTAGTTTCC 353 in the STT3A promoter. pBabe-PuroIkBalpha-mut (RRID:Addgene_15291; Addgene plasmid #15291, Watertown, MA, USA) [21] is a dominant-negative mutant NF-kB inhibitor (IkBaSR) and inhibits NF-kB activity.

Techniques: Glycoproteomics, Comparison, Variant Assay, Expressing, Cell Culture, Immunohistochemistry, Staining, Infection, Software, Western Blot, Knockdown, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro.

doi: 10.1016/j.ebiom.2021.103712

Figure Lengend Snippet: Figure 4. Impairment of STT3A compromises SARS-CoV-2 infectivity. (a) Luciferase assay was used to monitor the infection rate of pseudovirions generated from shSTT3A or shSTT3B HEK293T cells in HEK293T-ACE2 cells. Expression of spike in pseudovirus produced from shSTT3A and shSTT3B. Statistical method: one-way ANOVA, Tukey post hoc tests. **p < 0.01 (n = 3 with mean § SD shown). (b) Western blot analysis of N protein expression in A549-ACE2 cells infected with SARS-CoV-2 generated from control, shSTT3A or shSTT3B Vero E6 cells (left panel). N protein intensity was quantified by ImageJ (right panel). (c) Western blot analysis of spike and N protein expression in DMSO and NGI-1 treated SARS-CoV-2 infected Vero E6 cells. (d) Representative immunofluorescence images of N protein expression in DMSO and NGI-1 treated Vero E6 cell after SARS-CoV-2 infection. Scale bar, 100 mm. (e) SARS-CoV-2 neutralization IC50 of NGI-1 in Vero E6. Vero E6 cells was pretreated with NGI-1 1 h ahead at the indicated doses prior to SARS-CoV-2 infection (red). Cell viability of Vero E6 cells following NGI-1 treatment determined by MTT assay (black). Data shown are means § SD from representative triplicates. (f) Luciferase activity was measured at 48 h to determine SARS-CoV-2, Alpha, and Beta variants pseudoviral infectivity in DMSO and NGI-1 treated HEK293T-ACE2; Data shown are means § SD from represen- tative triplicates.

Article Snippet: The deleted NF-kB binding motif construct had a deletion at bases 363CGTAGTTTCC 353 in the STT3A promoter. pBabe-PuroIkBalpha-mut (RRID:Addgene_15291; Addgene plasmid #15291, Watertown, MA, USA) [21] is a dominant-negative mutant NF-kB inhibitor (IkBaSR) and inhibits NF-kB activity.

Techniques: Infection, Luciferase, Generated, Expressing, Produced, Western Blot, Control, Neutralization, MTT Assay, Activity Assay

Journal: PLoS ONE

Article Title: Crosstalks of the PTPIP51 interactome revealed in Her2 amplified breast cancer cells by the novel small molecule LDC3/Dynarrestin

doi: 10.1371/journal.pone.0216642

Figure Lengend Snippet: Antibody list.

Article Snippet: IκBα , Recombinant Human IκB alpha/NFKBIA protein 02 , Mouse monoclonal , MM02 , 1:100 , Sino Biological Inc., North Wales, PA, USA Cat.# 12045-H07E.

Techniques: Recombinant, Purification

Journal: Nature Communications

Article Title: Chronic activation of endothelial MAPK disrupts hematopoiesis via NFKB dependent inflammatory stress reversible by SCGF

doi: 10.1038/s41467-020-14478-8

Figure Lengend Snippet: a , b Immunoblot analysis and quantification demonstrating that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice does not affect ERK1/2 or p65 phosphorylation. Black arrowhead represents endogenous IκBα whereas red arrowhead represents IkB-SS transgene. c , d Representative immunofluorescence images and quantification demonstrating increased levels of nuclear p65 in BMECs derived from CDH5-MAPK mice as compared to controls. Note that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice ( CDH5-MAPK::IkB ) decreases nuclear p65 levels. Each dot within the bar graph represents nuclear p65 staining intensity per individual cell. e , f Heatmap and bar graph demonstrating increased expression of NF-κB-dependent inflammatory genes within BMECs of CDH5-MAPK mice. Crossing CDH5-MAPK with Tie2.IkB-SS mice ( CDH5-MAPK::IkB ) resulted in decreased expression of NF-κB-dependent target genes. Expression of Actb was used for normalization. Dendrograms represent unsupervised hierarchical clustering of the entire dataset ( n = 3 mice/cohort). Color scales represent relative mRNA abundance based on normalized gene expression. Raw data for Heatmaps included in Supplementary Table and Source Data. g Representative immunofluorescence images of femurs intravitally labeled with a vascular-specific CD144/VE-cadherin antibody (red) demonstrating that suppression of NF-κB signaling within endothelial cells of CDH5-MAPK mice resolves their vascular dilatation. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey’s correction was performed to determine significance. ** P < 0.01; *** P < 0.001; n.s. P > 0.05.

Article Snippet: IkB-SS expressing lentiviral vector was generated by sub-cloning the sequence of human IκBα super-suppressor (Addgene #15264) into pLVX Puro Vector (Clontech #632164).

Techniques: Western Blot, Expressing, Isolation, Phospho-proteomics, Immunofluorescence, Derivative Assay, Staining, Gene Expression, Labeling