superrepressor  (Addgene inc)

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: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of NF-kappa B pathway leads to deregulation of epithelial-mesenchymal transition and neural invasion in pancreatic cancer

doi: 10.1038/labinvest.2016.109

Figure Lengend Snippet: NF-κB activity is required for invasion: (a) Inhibition of NF-κB through IKBα repressor plasmid expression or BAY 11-7085 treatment decreased cellular invasiveness via Boyden chamber invasion assay; (b) BAY 11-7085 treatment decreased vimentin protein expression; expression of IKK plasmid rescues triptolide inhibition of (c) NF-κB activity; (d) EMT gene expression; (e) Boyden chamber invasion. Each bar is representative of three or more independent experiments; error bars are represented in SEM; and the asterisk (*) indicates a p value < 0.05.

Article Snippet: Plasmids: IKK (IKK-2 S177E, S191E) and IKBα (pBabe-Puro-IKBalpha-mut(superrepressor)) plasmids (Addgene).

Techniques: Activity Assay, Inhibition, Plasmid Preparation, Expressing, Invasion Assay

Journal: Oncotarget

Article Title: CD133 initiates tumors, induces epithelial-mesenchymal transition and increases metastasis in pancreatic cancer

doi:

Figure Lengend Snippet: (A) NF-κB activity correlated with CD133 expression and (B) In vitro invasion was decreased by NF-κB inhibition through IKK repression and pharmacological BAY 11–7085 treatment. (C) Decreased EMT genes upon IKBα repression in CD133 hi -MIA cells and (D) induction of NF-κB activity through IKK enhancer plasmid in EV-MIA control increased EMT related genes and conversely.

Article Snippet: IKK (IKK-2 S177E S191E) and IKBα (pBabe-Puro-IKBalpha-mut (superrepressor)) plasmids were obtained from Addgene.

Techniques: Activity Assay, Expressing, In Vitro, Inhibition, Plasmid Preparation, Control