Journal: The Journal of Clinical Investigation

Article Title: HIV latency is reversed by ACSS2-driven histone crotonylation

doi: 10.1172/JCI98071

Figure Lengend Snippet: (A) The J-Lat A1 cells were treated overnight with 30 mM Na-Cro (n = 3), 6 nM PEP005 (n = 3), 2 μM JQ1 (n = 4), 10 nM bryostatin (n = 4), 250 nM vorinostat (n = 3), 2 μM GSK343 (n = 4), 5 μM AZA-dC (n = 4), or 5 ng/ml PMA (n = 4) individually or in combination as indicated in the panel and evaluated for HIV reactivation by RT-qPCR. *P < 0.05 and ***P < 0.001 versus LRA alone. (B) The J-Lat A1 cells were pretreated with 250 nM of vorinostat; then 40 mM of Na-Cro was added overnight. The HIV reactivation was measured by real-time PCR. **P < 0.01 and ***P < 0.001 versus control treatment (n = 3). NS, not significant compared with Na-Cro treatment alone. (C) Effect of 30 mM Na-Cro, individually or in combination with PEP005, on reactivation of latent HIV was tested. The cells were treated overnight, and J-Lat A1 cells with HIV reactivation were measured by detection of GFP-expressing cells using flow cytometry. ***P < 0.001 versus control treatment; ###P < 0.001 versus PEP005 alone (n = 3). (D) U1 cells were treated similarly as in C; RT-qPCR was performed to measure viral expression. ***P < 0.001 vs. untreated control; ###P < 0.001 versus PEP005 treatment alone (n = 3). The data were analyzed with 1-way ANOVA.

Article Snippet: For reactivation of latent HIV, cells were treated with sodium crotonate (Na-Cro; Sigma-Aldrich), PEP005 (Tocris Bioscience), vorinostat (Santa Cruz Biotechnology), GSK343 (Sigma-Aldrich), or AZA-dC (Santa Cruz Biotechnology) for 4, 18, or 24 hours.

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Control, Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: HIV latency is reversed by ACSS2-driven histone crotonylation

doi: 10.1172/JCI98071

Figure Lengend Snippet: (A–C) Primary CD4+ T cells isolated from HIV-positive patients (n = 6) under ART were treated with 100 ng/ml PMA plus 2 μM ionomycin, 12 nM PEP005, 40 mM Na-Cro, or 12 nM PEP005 combined with 40 mM Na-Cro for 6 hours. Viral transcription from total RNA was analyzed by reverse transcriptase digital droplet PCR with primers targeting initiation (TAR region) (A), elongation (long LTR) (B), or full transcription [poly(A) region] (C) of the HIV genome. (D) Na-Cro synergistically reactivates the latent HIV with PEP005 in primary CD4+ T cells isolated from patients under suppressive ART. The difference between the observed fraction and predicted fraction or faxyo-faxyp (Δfaxy) was determined by Bliss independence analysis from 6 patient samples as described in the Supplemental Methods. **P < 0.01 and ***P < 0.001 versus faxyP (n = 6).

Article Snippet: For reactivation of latent HIV, cells were treated with sodium crotonate (Na-Cro; Sigma-Aldrich), PEP005 (Tocris Bioscience), vorinostat (Santa Cruz Biotechnology), GSK343 (Sigma-Aldrich), or AZA-dC (Santa Cruz Biotechnology) for 4, 18, or 24 hours.

Techniques: Isolation, Reverse Transcription

Journal: The EMBO Journal

Article Title: ER-phagy restrains inflammatory responses through its receptor UBAC2

doi: 10.1038/s44318-024-00232-z

Figure Lengend Snippet: ( A ) Lysates of THP-1 Mφs (left) or HT-29 cells (right) treated with TG (1 μM) for 1 h in the presence of Baf A1 (0.2 μM) were subjected to immunoprecipitation and immunoblot analysis. ( B ) Lysates of THP-1 Mφs (left) or HT-29 cells (right) cultured in EBSS for 3 h together with Baf A1 (0.2 μM) treatment were subjected to immunoprecipitation and immunoblot analysis. ( C ) Lysates of HeLa cells treated with EBSS for indicated time points in the presence of Baf A1 (0.2 μM) were subjected to immunoprecipitation and immunoblot analysis. ( D ) Immunoblot analysis of the knockdown efficiency of MARK2 by MARK2 -specific siRNAs in HeLa cells. ( E ) HeLa cells were transfected with scramble or MARK2 -specific siRNA and treated with EBSS for 3 h in the presence of Baf A1 (0.2 μM). The lysates were subjected to immunoprecipitation and immunoblot analysis. ( F ) WT or MARK2 KO THP-1 Mφs were cultured in EBSS for 3 h with the existence of Baf A1 (0.2 μM). The lysates were treated with 2 mM disuccinimidyl suberate (DSS) cross-linker or immunoprecipitated using phosphoserine/threonine/tyrosine polyclonal antibody, and detected by immunoblot. ( G , H ) WT or MARK2 KO HT-29 cells were treated with TG (1 μM) for 1 h ( G ) and cultured in EBSS for 3 h ( H ) in the presence of Baf A1 (0.2 μM). The lysates were treated with 2 mM disuccinimidyl suberate (DSS) cross-linker or immunoprecipitated using phosphoserine/threonine/tyrosine polyclonal antibody, and detected by immunoblot. ( I ) 293T cells were transfected with scramble or MARK2 -specific siRNA and then transfected with vector of WT or S223A UBAC2. After incubation the cells with EBSS for 3 h in the presence of Baf A1 (0.2 μM), the lysates were harvested for immunoprecipitation and immunoblot analysis. ( J ) The band intensities of RFP and RFP-GFP in the similar samples as Fig. from three independent experiments performed in duplicate were quantified and the ratios of RFP/RFP-GFP were shown. ( K ) HeLa cells stably expressing the ER-phagy reporter were transfected with scramble or MARK2 -specific siRNA alongside with WT or mutated UBAC2 plasmids. The cells were then cultured in the presence of doxycycline for 24 h to induce the reporter and the protein was harvested for immunoblot analysis. ( L ) The band intensities of RFP and RFP-GFP in the similar samples as ( K ) from three independent experiments performed in duplicate were quantified and the ratios of RFP/RFP-GFP were shown. ( M ) HeLa cells stably expressing the ER-phagy reporter were transfected with scramble or UBAC2 -specific siRNA and then treated with PEP005 with indicated dosages for 1 h. The cells were cultured in EBSS for 3 h and the lysates were harvested for immunoblot analysis. Data information: For ( A – I , K , M ), one representative experiment out of three was shown. In ( J , L ), data are presented as the mean ± SEM of three independent biological experiments. The statistical significance of the difference was analyzed by unpaired two-tailed Student’s t test, and the P values were shown.

Article Snippet: Carfilzomib (cat. HY-10455), tunicamycin (cat. HY-A0098) and PEP005 (cat. HY-B0719) were purchased from MedChemExpress (MCE).

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Knockdown, Transfection, Plasmid Preparation, Incubation, Stable Transfection, Expressing, Two Tailed Test