Journal: Science Advances

Article Title: Histone decrotonylation plays a distinct role in HIV latency

doi: 10.1126/sciadv.aec0149

Figure Lengend Snippet: ( A ) Primary CD4 + T cell model of latency was activated with anti-CD3/CD28 Dynabeads. The percentage of GFP + cells was measured by flow cytometry (top). The levels of the ncNF-κB components p100 and p52, as well as ENL and CDYL were measured by Western blot (bottom) ( n = 3). The relative intensity was shown below with results normalized to dimethyl sulfoxide (DMSO) control. MW, molecular weight. ( B ) Primary CD4 + T cells from HIV-negative donors were infected with HIV-1 (SF162) and treated with or without 40 mM NaCr ( n = 3). HIV transcription levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting the HIV LTR, with results normalized to day 1 (left). The percentage of p24 + cells was measured by HIV-Flow assay (right). ( C ) TZM-bl luciferase reporter cells were transfected with empty vector (EV), Tat, wild-type (WT) HDAC3, or mutant HDAC3 (HDAC3-VRPP or HDAC3-Y298H), or Tat in combination with WT or mutant HDAC3 for 2 days ( n = 3). ( D ) A similar luciferase assay was performed with WT p300 and mutant p300-I1395G. Tat-induced HIV transcription was measured using a luciferase assay. Results are expressed as relative light units (RLU) and normalized to the EV control ( n = 4). The P values were determined using two-way analysis of variance (ANOVA) with multiple comparisons [(B), left], two-tailed unpaired Student’s t test [(B), right], or one-way ANOVA with multiple comparisons (C). Error bars represent SD; * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: We first confirmed that 1 to 20 nM YX968 (MedChemExpress) selectively degraded HDAC3 and HDAC8, but not HDAC1 and HDAC2, in 2D10 cells, unless the dosage was higher than 50 nM (fig. S5D).

Techniques: Flow Cytometry, Western Blot, Control, Molecular Weight, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Two Tailed Test

Journal: Science Advances

Article Title: Histone decrotonylation plays a distinct role in HIV latency

doi: 10.1126/sciadv.aec0149

Figure Lengend Snippet: ( A ) A modified version of the HDAC3 crystal structure (PDB: 4A69) with the DAD and IP 4 removed compared with the AF3 predicted HDAC3 crystal structure. ( B ) Docking the selected HDACis into the enzyme pocket of HDAC3. ( C and D ) Primary CD4 + T cells were treated with the indicated HDACis for 0.5 hours. Total histone H3 and histone PTMs were detected by Western blot. The relative intensity was shown below with results normalized to DMSO control. HDAC3/NCOR1 (0.3 mg/ml) was incubated with crotonylated histone H3 ( E ) or acetylated histone H3 ( F ) in the presence or absence of different concentrations of citarinostat. Total histone H3 and histone PTMs were detected by Western blot. For (C) and (D), DMSO was used as a negative control. Results are representative of three biological replicate experiments with consistent results.

Article Snippet: We first confirmed that 1 to 20 nM YX968 (MedChemExpress) selectively degraded HDAC3 and HDAC8, but not HDAC1 and HDAC2, in 2D10 cells, unless the dosage was higher than 50 nM (fig. S5D).

Techniques: Modification, Western Blot, Control, Incubation, Negative Control

Journal: Science Advances

Article Title: Histone decrotonylation plays a distinct role in HIV latency

doi: 10.1126/sciadv.aec0149

Figure Lengend Snippet: Structural overlay of HDAC1, HDAC2, HDAC3, and HDAC8 bound to SAHA ( A ) and citarinostat ( B ). HDAC1, green; HDAC2, cyan; HDAC3, wheat; HDAC8, pink. HDAC2 (0.05 mg/ml) ( C ) or HDAC8 (0.4 mg/ml) ( D ) were incubated with crotonylated histone H3 or acetylated histone H3 in the presence or absence of different concentrations of citarinostat. Total histone H3 and histone PTMs were detected by Western blot. ( E ) HDAC3 was knocked out in 2D10 cells via CRISPR-Cas9, using a nontargeting guide RNA as the control, and the cells were cultured for 5 days. NT, nontargeting control; KO, knock out. ( F ) In a separate experiment, 2D10 cells were pretreated for 24 hours with DMSO or 20 nM YX968, an HDAC3/8 dual PROTAC. For both experimental series, cells were subsequently treated for 30 min with the following HDACis: DMSO (vehicle), 350 nM SAHA, 0.1 μM citarinostat, 5 μM RGFP966, or 5 μM mod-RGFP966. Histones were then extracted and analyzed by Western blot for specific PTMs and total histone H3 levels.

Article Snippet: We first confirmed that 1 to 20 nM YX968 (MedChemExpress) selectively degraded HDAC3 and HDAC8, but not HDAC1 and HDAC2, in 2D10 cells, unless the dosage was higher than 50 nM (fig. S5D).

Techniques: Incubation, Western Blot, CRISPR, Control, Cell Culture, Knock-Out

Journal: Science Advances

Article Title: Histone decrotonylation plays a distinct role in HIV latency

doi: 10.1126/sciadv.aec0149

Figure Lengend Snippet: ( A ) Citarinostat, RGFP966, and mod-RGFP966 were docked against the artificial intelligence–generated enzyme pocket of HDAC3. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; Q, Gln; R, Arg. ( B ) Primary CD4 + T cells were nucleofected with 1 μg of EV or HDAC3 WT/D57A/Q113A/R265A/R301A. After 5 to 7 days, total histone H3 and histone PTMs were detected by Western blot. The relative intensity was shown below with results normalized to WT HDAC3. PanKbu, butyryllysine; PanKlac, L-lactyllysine; PanKpr, propionyllysine.

Article Snippet: We first confirmed that 1 to 20 nM YX968 (MedChemExpress) selectively degraded HDAC3 and HDAC8, but not HDAC1 and HDAC2, in 2D10 cells, unless the dosage was higher than 50 nM (fig. S5D).

Techniques: Generated, Western Blot

Journal: Science Advances

Article Title: Histone decrotonylation plays a distinct role in HIV latency

doi: 10.1126/sciadv.aec0149

Figure Lengend Snippet: Structural overlay of HDAC1, HDAC2, HDAC3, and HDAC8 bound to SAHA ( A ) and citarinostat ( B ). HDAC1, green; HDAC2, cyan; HDAC3, wheat; HDAC8, pink. HDAC2 (0.05 mg/ml) ( C ) or HDAC8 (0.4 mg/ml) ( D ) were incubated with crotonylated histone H3 or acetylated histone H3 in the presence or absence of different concentrations of citarinostat. Total histone H3 and histone PTMs were detected by Western blot. ( E ) HDAC3 was knocked out in 2D10 cells via CRISPR-Cas9, using a nontargeting guide RNA as the control, and the cells were cultured for 5 days. NT, nontargeting control; KO, knock out. ( F ) In a separate experiment, 2D10 cells were pretreated for 24 hours with DMSO or 20 nM YX968, an HDAC3/8 dual PROTAC. For both experimental series, cells were subsequently treated for 30 min with the following HDACis: DMSO (vehicle), 350 nM SAHA, 0.1 μM citarinostat, 5 μM RGFP966, or 5 μM mod-RGFP966. Histones were then extracted and analyzed by Western blot for specific PTMs and total histone H3 levels.

Article Snippet: We first confirmed that 1 to 20 nM YX968 (MedChemExpress) selectively degraded HDAC3 and HDAC8, but not HDAC1 and HDAC2, in 2D10 cells, unless the dosage was higher than 50 nM (fig. S5D).

Techniques: Incubation, Western Blot, CRISPR, Control, Cell Culture, Knock-Out

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Application of 18 β -glycyrrhetinic acid Fluorescent probes in cell imaging

doi: 10.1080/14756366.2026.2631869

Figure Lengend Snippet: Effects of 18 β -GA-PAL-ABP on LPS-induced release of HDAC8, STAT3, P-STAT3, and SOCS3. A: expression of HDAC8 protein; B: expression of STAT3 protein; C: expression of P-STAT3 protein; and D: expression of SOCS3 protein. E: The protein expression map in RAW 264.7 cells treated with Class I compounds and drug concentrations; F: The protein expression map in RAW 264.7 cells treated with Class II compounds and drug concentrations; G: The protein expression map in RAW 264.7 cells treated with Class III compounds and drug concentrations. Compared to the Control group, # p < 0.05, ## p < 0.01, ### p < 0.001. Compared to the Model group, * p < 0.05, ** p < 0.01, *** p < 0.001. Compared to the 18β-GA group, + p < 0.05, ++ p < 0.01, +++ p < 0.001.

Article Snippet: GAPDH (8884, CST, 1:5000), HDAC8 (#66042, CST, 1:1500), P-STAT3 (#9154, CST, 1:1500), STAT3 (#12640, CST, 1:1000), SOCS3 (#52113, CST, 1:2000), SDS-PAGE reagents (M00657, Genescript, Nanjing, China).

Techniques: Expressing, Control