Journal: Genes & Development

Article Title: Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance

doi: 10.1101/gad.349585.122

Figure Lengend Snippet: Senescence-induced canonical NF-κB signaling in endothelial cells regulates downstream signaling and lymphocyte recruitment. ( A ) Experimental setup: direct coculture of growing or RIS ER:HRAS G12V IMR90 cells (asterisks) with HUVECs (arrowheads) expressing the IκBα superrepressor (SR) or vector control. ( B ) Representative immunofluorescence of coculture with senescence-dependent IL8 expression in both CD31 − IMR90s and CD31 + HUVECs. n = 5 biological replicates. Scale bar, 30 µm. ( C ) Separate quantification of IL8 positivity from the two cell types. Dots are individual replicates, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test; (****) P ≤ 0.0001. ( D ) Experimental setup: HUVECs expressing the SR or vector control were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells for 16 h before harvesting for flow cytometry for ICAM1 ( middle panel) and ICOSLG ( right panel). n ≥ 3 biological replicates. ( E ) Experimental setup: HUVECs were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells with vehicle or the indicated NF-κB inhibitors for 16 h before harvesting for flow cytometry for ICAM1. n ≥ 3 biological replicates. ( F ) Experimental setup: LSECs or HUVECs with the indicated genetic or pharmacological NF-κB inhibitors were incubated in CM from growing or RIS ER:HRAS G12V IMR90 cells for 16 h before performing a flow adhesion assay and analyses of lymphocyte adherence and trans -endothelial migration. ( G ) Representative photomicrographs of HUVECs with the indicated constructs and CM, showing adherent lymphocytes (black arrows). ( H ) Trans -endothelial migration of lymphocytes in the indicated cell lines and conditions. Dots are individual replicates, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test; (**) P ≤ 0.01, (****) P ≤ 0.0001.

Article Snippet: The following retroviral vectors were used: pLNCX2 ER:HRAS G12V (Addgene 67844; RRID: Addgene_67844) , pBabe-empty vector , and pBabe-IκBα S32A/S36A -“superrepressor” (a gift from William Hahn; Addgene 15291; RRID: Addgene_15291).

Techniques: Expressing, Plasmid Preparation, Immunofluorescence, Incubation, Flow Cytometry, Cell Adhesion Assay, Migration, Construct

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