Journal: iScience

Article Title: Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52

doi: 10.1016/j.isci.2021.103649

Figure Lengend Snippet: Crotonylation stimulated p100 cleavage into p52 in ncNF-κB pathway in CD4+ T cells (A) Model of ncNF-kB signaling pathway and the site of action of IAPi/SMACm. (B) 2D10 cells were treated with 1 nM AZD5582 (AZD), 40 mM NaCr or their combination. Cells were harvested 24 h posttreatment and total proteins were prepared for Western blot. (C) J-Lat A1 cells were treated with 1 nM AZD, 40 mM NaCr or their combination. U1 cells were treated with 5 nM AZD, or 20 mM NaCr or their combination while J-Lat 10.6 cells were treated with 10 nM AZD, or 40 mM NaCr or their combination. Primary CD4+ T cells were treated with 100 nM AZD, or 30 mM NaCr or their combination. Cells were harvested 24 h posttreatment and total proteins were prepared for the examination of p100/52 protein cleavage by Western blot. (D) U1 or 2D10 cells were treated as in (C). Then, nucleus and cytoplasm were fractionated and subjected for Western blot analysis, where actin and β-TrCP served as cytoplasmic protein controls and HADC1 or TRIM27 served as nuclear protein controls. Data are represented as mean ± SEM.

Article Snippet: TRIM27 , Applied Biosystems , Hs01100632_ml.

Techniques: Western Blot

Journal: iScience

Article Title: Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52

doi: 10.1016/j.isci.2021.103649

Figure Lengend Snippet: TRIM27 is involved in HIV transcription (A–C) TRIM27 was knocked down in 2D10 cells after transfection of control siRNA or TRIM27 siRNA. Two days after knockdown, the cells were treated with AZD5582 (AZD) overnight. Then, the cells were harvested and TRIM27 and HIV RNA expression was analyzed by qPCR. ∗, p < 0.05; ∗∗∗, p < 0.001, analyzed by two-tailed T test (n = 3–5). Data are represented as mean ± SEM. (D–F) TRIM27 was knocked down in the primary CD4+ T cell model of latency after transfection of control siRNA or TRIM27 siRNA. Two days after knockdown, the cells were treated with AZD5582 (AZD) overnight. Then, the cells were harvested and TRIM27 and HIV RNA expression was analyzed by qPCR (F). ∗, p < 0.05, analyzed by two-tailed T test (n = 3). Data are represented as mean ± SEM.

Article Snippet: TRIM27 , Applied Biosystems , Hs01100632_ml.

Techniques: Transfection, Control, Knockdown, RNA Expression, Two Tailed Test

Journal: iScience

Article Title: Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52

doi: 10.1016/j.isci.2021.103649

Figure Lengend Snippet: TRIM27 interacts with USP7 to upregulate HIV transcription (A) 2D10 cells were treated with or without 40 mM NaCr for 24 h. Cells were harvested for immunoprecipitation by anti-TRIM27 antibody, followed by Western blot with anti-TRIM27 antibody and anti-USP7 antibody. (B) 2D10 cells were treated with 1 nM AZD5582 (AZD), 30 mM NaCr, 5 μM USP7 inhibitor (USP7i) P5091 or their combinations for 24 h. Cells were harvested and total RNA were isolated for HIV RNA expression analyses by qPCR (n = 3). ∗∗∗, p < 0.001, analyzed by One-way ANOVA test. Data are represented as mean ± SEM. (C) 2D10 cells were treated as in B and total proteins were prepared to examine ncNF-κB related protein expression.

Article Snippet: TRIM27 , Applied Biosystems , Hs01100632_ml.

Techniques: Immunoprecipitation, Western Blot, Isolation, RNA Expression, Expressing

Journal: iScience

Article Title: Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52

doi: 10.1016/j.isci.2021.103649

Figure Lengend Snippet:

Article Snippet: TRIM27 , Applied Biosystems , Hs01100632_ml.

Techniques: Recombinant, Extraction, Lysis, Protease Inhibitor, Reverse Transcription, Cell Isolation, Staining, Binding Assay, Control, Software