nuclei rabbit nrf2 antibody  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

doi: 10.1038/s41467-024-48422-x

Figure Lengend Snippet: a NRF2 protein levels in control and NRF2 knockout (KO) 786-O cells treated with 4-OI (75 μM) for 24 h using confocal microscopy. Blue: actin filaments, green: NRF2. Scale bars, 50 μm. b KEAP1 levels analyzed in 786-O cells treated with alkynated 4-OI (4-OI-alk) (125 μM) for 4 or 24 h with or without non-alkynated 4-OI (125 μM) by anti-KEAP1 immunoblotting. c Viral RNA content assessed by RNA sequencing in VSVΔ51-infected (MOI of 0.01) control and NRF2 KO 786-O cells with or without 4-OI (75 μM) pretreatment. d – f Immunoblot analysis in control and NRF2 KO cells pretreated with 4-OI (75 μM) before VSVΔ51 challenge (MOI of 0.01) ( d ). Fluorescence microscopy showing VSVΔ51-RFP spread and cellular layer integrity with Hoechst stain overlay (Scale bars, 300 μm) ( e ) and quantification of infected cells by flow cytometry at 17 h post-infection ( f ). g – j Quantification of virus-infected cells by flow cytometry in 786-O cells transiently KO for NRF2 ( g ) or KEAP1 ( j ). Immunoblot analysis in NRF2 KO ( h ) and KEAP1 KO cells ( i ) pretreated with 4-OI (75 μM) before VSVΔ51 challenge (MOI of 0.01). k 786-O cells incubated with L-NAC (1 mM) for 3 h before 4-OI challenge (75 μM) for 24 h, then infected with VSVΔ51-RFP (MOI of 0.01). Quantification of infected cells by flow cytometry at 17 h post-infection. Data are means ± SEM from two independent experiments in biological duplicates and triplicates in ( f ), and in biological triplicates and quadruplicates in ( g ). Two experiments in biological quadruplicates in ( j ) and ( k ). Images from one representative experiment in triplicates in ( a ) and ( e ). Data in ( b ) and ( d ) from one representative of three independent experiments. Data as means ± SEM from one experiment in biological triplicates in ( c ). Statistics indicate significance by one-way ANOVA for ( f , g , j , k ). Vertical stacks of bands are not derived from the same membrane in ( d , h , I ). Source data provided in the Source Data file.

Article Snippet: To show NRF2 and P65 translocation into the nuclei rabbit NRF2 antibody (12721, Cell Signaling, 1:200) or rabbit-cleaved P65 antibody (8242, Cell Signaling, 1:200) were applied respectively.

Techniques: Control, Knock-Out, Confocal Microscopy, Western Blot, RNA Sequencing, Infection, Fluorescence, Microscopy, Staining, Flow Cytometry, Virus, Incubation, Derivative Assay, Membrane

Journal: Nature Communications

Article Title: Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

doi: 10.1038/s41467-024-48422-x

Figure Lengend Snippet: a – c 786-O cells pretreated with 4-OI (75 μM) for 24 h and infected with VSVΔ51 (MOI 0.01) for 17 h. RNA sequencing analysis emphasizing on antiviral genes (blue dots) in the volcano plot ( a ), differentially expressed interferon-stimulated genes (ISGs) in the heat map ( b ), and top KEGG pathways affected by 4-OI during viral infection (one-sided hypergeometric test, Benjamini–Hochberg method was applied to adjust the p -value for multiple testing) ( c ). d , e 786-O cells pretreated with 4-OI (125 μM) for 24 h and infected with VSVΔ51-RFP (MOI 0.01) for 24 h. IFIT1 levels assessed by fluorescence microscopy ( d ), and Western blot performed on cell lysates for antiviral proteins ( e ). f 786-O cells pretreated with 4-OI (75 μM) for 24 h infected with wild-type VSV (wt VSV) or VSVΔ51 at MOI 0.01. Viral titers determined 24 h post-infection. g – i Control and NRF2 KO 786-O cells, as well as 786-O cells transiently KO for NRF2 or KEAP using CRISPR/Cas9, pretreated with 4-OI (75 μM) for 24 h and infected with VSVΔ51 (MOI 0.01) for 17 h. Immunoblots in ( g , h ). CXCL10 release measured by ELISA from supernatants in ( I ). j Control and NRF2 KO 786-O cells pretreated with 4-OI (75 μM) for 24 h and stimulated with the RIG-I agonist M8 (3.5 ng/mL) for 5 h. Western blot performed on cell lysates. Data are from one experiment performed in triplicate in ( a – c ). Images are from one experiment in ( d ). Data are from one representative experiment performed at least three times in ( e ). Data are depicted as means ± SEM from one experiment performed in triplicate in ( f ). Data are from one representative experiment out of three in ( g ), out of two in ( h ) and ( j ). Data are depicted as means ± SEM from two experiments performed in triplicate in ( I ). Statistics indicate significance by two-way ANOVA for ( f , I ). Vertical stacks of bands are not derived from the same membrane in ( e , g , h , j ). Source data provided as a Source Data file.

Article Snippet: To show NRF2 and P65 translocation into the nuclei rabbit NRF2 antibody (12721, Cell Signaling, 1:200) or rabbit-cleaved P65 antibody (8242, Cell Signaling, 1:200) were applied respectively.

Techniques: Infection, RNA Sequencing, Fluorescence, Microscopy, Western Blot, Control, CRISPR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Membrane

Journal: Nature Communications

Article Title: Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

doi: 10.1038/s41467-024-48422-x

Figure Lengend Snippet: a CiiiDER analysis to identify overrepresented transcription factor binding sites in 786-O cells treated or not with 4-OI (75 µM) prior VSVΔ51 (MOI 0.01 for 17 h). b , c Confocal microscopy of p65 nuclear translocation in 786-O cells treated or not with 4-OI following VSVΔ51 (Scale, 20 μm) ( b ); IL-6 levels measured by ELISA ( c ). d , e Data from Runtsch et al. . THP1 cells treated with 4-OI following IA-DTB, cell lysis, and LC-MS measurements of modified cysteines. f 786-O cells upon 4-OI-alk with or without 4-OI (both 125 µM). Immunoblotting of IKKβ, IKKγ, and IKK ε before and after biotin enrichment. g Illustration of 4-OI binding (green) to IKKβ at Cys179 (left) and 2D ligand-protein interactions (right). Lipophilicity protein surface: lipophilic (cyan), hydrophilic (violet), neutral (white), α-helices (cyan), β-sheets (yellow), loops (cyan). h Luciferase NF-kB promotor activity in control and NRF2 KO 786-O treated or not with 4-OI prior to control (pc) or IKKβ plasmid transfection. i Immunoblotting of IKKα/β, IκBα, and P65 phosphorylation in 786-O cells treated or not with 4-OI prior VSVΔ51. j , k IKKβ KO 786-O cells treated or not with 4-OI following VSVΔ51, immunoblotting ( j ) and flow ( k ) analyses. l Luciferase assay of NF-κB promotor activity in HEK293 cells stimulated or not with 4-OI prior to IKKβ wt or IKKβ C179A plasmid transfection. Data from one representative experiment in ( a ), two independent experiments in ( b ). Data are depicted as means ± SEM from two experiments in triplicates in ( c ), three independent experiments in ( d , e ). Data from one representative experiment out of three in ( f ). Data are depicted as means from one experiment in duplicates in ( h ). Data from one representative experiment performed twice in ( I , j ). Data are the means ± SEM from two experiments performed in quadruplicates in ( k ). Data are the means ± SEM from two experiments in triplicates in ( l ). Statistical significance by two-way ANOVA for ( c , l ) and one-way ANOVA for ( k ). Vertical stacks of bands are not derived from the same membrane in ( f , i , j ). Source data are provided in Source Data file.

Article Snippet: To show NRF2 and P65 translocation into the nuclei rabbit NRF2 antibody (12721, Cell Signaling, 1:200) or rabbit-cleaved P65 antibody (8242, Cell Signaling, 1:200) were applied respectively.

Techniques: Binding Assay, Confocal Microscopy, Translocation Assay, Enzyme-linked Immunosorbent Assay, Lysis, Liquid Chromatography with Mass Spectroscopy, Modification, Western Blot, Luciferase, Activity Assay, Control, Plasmid Preparation, Transfection, Phospho-proteomics, Derivative Assay, Membrane