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: bioRxiv

Article Title: Mitochondrial carrier SLC25A34 links clock, diet, and temperature control of interorganellar lipid cycling

doi: 10.64898/2026.05.30.724257

Figure Lengend Snippet: A, schematic of the multi-omic approach to discover Slc25a34 ; comparing the proteome of BAT from (1) cold-exposed mice (top left, 3 weeks cold exposure vs thermoneutrality, FC>2) and (2) Rev-erbα WKO mice (bottom left, Rev-erbα WKO vs Control, FC>2), (3) cistrome of BAT from Control and Rev-erbα WKO mice (top right, REV-ERBα binding sites located within 10 kb of the nearest gene that show higher occupancy in cold conditions than at thermoneutrality), and (4) BAT transcriptome from cold-exposed Rev-erbα/β dBKO mice (bottom right, genes induced by cold in control mice (FC > 2, FDR < 0.05) and genes induced by double Rev-erbα/β knockout in BAT at thermoneutrality (FC > 2, FDR < 0.05). B , Slc25a34 expression across tissues, fold change relative to the expression level in the tissue at 29°C. C , Slc25a34 and Slc25a35 mRNA and, D , protein levels in BAT during cold adaptation, significance indicates effects of cold exposure compared to 29°C. E , Slc25a34 mRNA levels in scWAT during cold adaptation, significance indicates effects of cold exposure compared to 29°C. F , Circadian expression of Slc25a34 and Slc25a35 mRNA levels in control and Rev-erbα WKO mice acclimated to 29°C. G , SLC25A34 and SLC25A35 protein in BAT of control and Rev-erbα WKO mice at 22°C at ZT10. H , Slc25a34 mRNA levels in BAT of control and Rev-erbα WKO mice at 29°C and after 1.5h of 4°C exposure (ZT4 – ZT5.5). I , REV-ERBα occupancy in the proximal Slc25a34 promoter in BAT at 29°C and after 6 h of 4°C exposure of control and Rev-erbα WKO mice (ZT10). J , Slc25a34 mRNA levels in BAT from control and Hdac3 AKO mice at 22°C and after 3h at 4°C. K , Schematic depicting the role of REV-ERBα in coordinating circadian and cold-mediated regulation of Slc25a34 expression. For all panels, data are represented as mean ±SEM, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****, unpaired two-tailed multiple t-tests (B, H), One-way ANOVA with the Benjamini, Krieger, and Yekutieli two-stage linear step-up procedure for multiple comparisons correction (C-E), Two-way ANOVA with the Benjamini, Krieger, and Yekutieli two-stage linear step-up procedure for multiple comparisons correction (F, G, J).

Article Snippet: To generate the mouse strain with the adipose-specific Hdac3 knockout ( Hdac3 AKO), Hdac3 f/f mice maintained on a C57BL/6 background were bred to B6;FVB-Tg(Adipoq-Cre)1Evdr/J mice form Jackson laboratory (stock No: 010803) ( ).

Techniques: Control, Binding Assay, Knock-Out, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Mitochondrial carrier SLC25A34 links clock, diet, and temperature control of interorganellar lipid cycling

doi: 10.64898/2026.05.30.724257

Figure Lengend Snippet: A , Slc25a35 expression across tissues, fold change relative to the expression level in the tissue at TN. B , Slc25a35 mRNA levels in scWAT during cold adaptation. C , Slc25a35 mRNA levels in BAT of control and Rev-erbα KO mice at TN and cold. D , Slc25a35 mRNA levels in BAT from control and Hdac3 AKO mice at TN and after 6 h of cold exposure (ZT10). For all panels, error bars represent ±SEM, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****, unpaired two-tailed multiple t-tests (A), One-way ANOVA (B) and Two-way ANOVA (C, D) with the Benjamini, Krieger, and Yekutieli two-stage linear step-up procedure for multiple comparisons correction.

Article Snippet: To generate the mouse strain with the adipose-specific Hdac3 knockout ( Hdac3 AKO), Hdac3 f/f mice maintained on a C57BL/6 background were bred to B6;FVB-Tg(Adipoq-Cre)1Evdr/J mice form Jackson laboratory (stock No: 010803) ( ).

Techniques: Expressing, Control, Two Tailed Test

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: HDAC3 is upregulated in microglia of the spinal cord from EAE model mice. A , B The protein levels of HDAC3 and GAPDH in the spinal cord from ctrl ( n = 4) and EAE ( n = 4) mice were determined by western blot ( A ) and the gray values were analyzed by Image J ( B ). C , D The mRNA ( n = 6 for each group) and protein ( n = 3 for each group) levels of HDAC3 in primary microglia isolated from Ctrl and EAE mice were determined by real-time PCR ( C ) and western blot ( D ). E , F The protein levels of HDAC3 in Iba1 positive cells in the spinal cord from ctrl and EAE mice were determined by immunostaining ( E ) and the fluorescence intensity were quantified by Image J ( F ). G – I The protein levels of HDAC3 in CD11b + CD45 low cells in the spinal cord from ctrl ( n = 4) and EAE ( n = 4) mice were determined by flow cytometry ( G , H ) and the mean fluorescent intensity of HDAC3 in CD11b + CD45 low cells were analyzed ( I ) (*indicates p < 0.05, **indicates p < 0.01 by Student’s t -test)

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: Western Blot, Isolation, Real-time Polymerase Chain Reaction, Immunostaining, Fluorescence, Flow Cytometry

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: Mice with microglial Hdac3-deficiency exhibited higher sensitivity to EAE induction. A Spinal cord slices from WT and Hdac3 cKO mice were stained with anti-HDAC3 (red) and anti-Iba1 (green) antibody, and the yellow arrow pointed to the signal of HDAC3 responding to the position of the Iba1-positive area. B The protein levels of HDAC3 and GAPDH in the primary microglia isolated from WT ( n = 3) and Hdac3 cKO ( n = 3) mice were determined by western blot and the gray values were analyzed by Image J. C Clinical score of WT EAE ( n = 8) and Hdac3 cKO EAE ( n = 8) mice were recorded every day post-immunization. D , E Spinal cord slices from WT EAE ( n = 6) and Hdac3 cKO EAE ( n = 6) mice were stained with Fast-blue and eosin ( D ), and the percentage of the Fast-blue negative area ( E ) was analyzed by ImageJ. F Spinal cord slices from WT EAE and Hdac3 cKO EAE mice were stained with anti-MBP antibody. G , H Spinal cord slices from WT EAE ( n = 7) and Hdac3 cKO EAE ( n = 7) mice were stained with H/E staining ( G ), and the number of infiltrated cells was counted manually ( H ). (*indicates p < 0.05, **indicates p < 0.01 by ANOVA or Student’s t -test)

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: Staining, Isolation, Western Blot

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: Microglial Hdac3-deficiency promoted periphery immune infiltration in the spinal cord during EAE development. A , B Kyoto encyclopedia of genes and genomes (KEGG) analysis ( A ) and ImmunoSystemProcess analysis ( B ) of the different expressed genes (DEGs) between the spinal cord from WT EAE mice and Hdac3 cKO EAE mice in RNA-sequencing data were performed by Cytoscape. C The mRNA levels of marker genes for T cell, B cell, NK cell and granulocyte in RNA-sequencing data from WT EAE mice and Hdac3 cKO EAE mice were displayed as heatmap. D , E Gene set enrichment analysis (GSEA) showed that the TCR signaling pathway ( D ) and positive regulation of leukocyte migration ( E ) were upregulated in the spinal cord from Hdac3 cKO EAE mice. F , G Genes involved in the TCR signaling pathway ( F ) and positive regulation of leukocyte migration ( G ) were upregulated in the spinal cord from Hdac3 cKO EAE mice ( N = 3 for each group)

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: RNA Sequencing, Marker, Migration

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: Inhibition of HDAC3 increased CD8 + T cell infiltration in spinal cord during EAE development. A – C The mRNA levels of CD3g ( A ), CD8a ( B ) and FasL ( C ) in the spinal cord from WT Ctrl ( n = 4), EAE mice administrated with RGFP966 ( n = 5) or vehicle ( n = 5) were determined by real-time PCR. D – F The mRNA levels of CD3g ( D ), CD8a ( E ) and FasL ( F ) in the spinal cord from WT Ctrl ( n = 6), WT EAE ( n = 6) and Hdac3 cKO EAE ( n = 6) mice were determined by real-time PCR. G – I The number of CD8 + T cell in the spinal cord from WT Ctrl, EAE mice administered with RGFP966 ( n = 6) or vehicle ( n = 6) ( G , H ), and WT EAE ( n = 5) and Hdac3 cKO EAE ( n = 5) mice ( G and I ) were detected by immunostaining with anti-CD8a and the number of CD8a-positive cells were counted. J , K The percentage of CD8 + T cells (gated in CD8a and CD3e double positive) in the spinal cord from WT Ctrl ( n = 6), WT EAE ( n = 6) and Hdac3 cKO EAE ( n = 6) mice were analyzed by flow cytometry with antibodies against CD8a and CD3e. (*indicates p < 0.05, **indicates p < 0.01 by ANOVA or Student’s t -test)

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Immunostaining, Flow Cytometry

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: Inhibition of HDAC3 increased the expression of CCL5 in spinal cord during EAE development. A Scatter diagram of the fold_change and p _value of the mRNA levels of chemokines between WT and Hdac3 cKO primary microglia analyzed by RNA-sequencing. B The chemokines that upregulated in Hdac3 cKO EAE spinal cord and Hdac3 cKO primary microglia were analyzed by RNA-sequencing. C , D The mRNA levels of CCL5 in the spinal cord from Ctrl ( n = 4), EAE mice administered with RGFP966 ( n = 7) or vehicle ( n = 5) ( C ), and WT_EAE ( n = 6) and Hdac3 cKO_EAE ( n = 6) mice ( D ) were determined by real-time PCR. E , F The mRNA levels of CCL5 ( E ) and HDAC3 ( F ) in WT ( n = 3) and Hdac3 cKO ( n = 3) primary microglia were determined by real-time PCR. G – I The protein levels of CCL5 and Iba1 in the spinal cord from Ctrl, EAE mice administrated with RGFP966 ( n = 6) or vehicle ( n = 6) ( G , H ), and WT_EAE ( n = 6) and Hdac3 cKO_EAE ( n = 6) mice ( G and I ) were detected by immunostaining with anti-CCL5 and anti-Iba1 antibodies and the number of CCL5 + Iba1 + cells were counted. (*indicates p < 0.05, **indicates p < 0.01 by ANOVA or Student’s t -test)

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: Inhibition, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Immunostaining

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: Microglial HDAC3 restrains IFN-γ induced expression of CCL5 by deacetylating on histone 3 lysine 9. A , B The mRNA levels ( A ) and protein levels ( B ) of HDAC3 and β-actin in N9 cells transfected with siRNA against HDAC3 (siHDAC3) or negative control (siNC) were determined by real-time PCR 72 h post-transfection. C , D N9 cells were pretreated with siRNA ( C ) or RGFP966 ( D ) as above, and then were stimulated with rIFN-γ (10 ng/ml) for indicating hours, and the cells were collected for detecting the mRNA levels of CCL5 by real-time PCR. E , F N9 cells were pretreated with siRNA ( E ) or RGFP966 ( F ) as above, and then were stimulated with rIFN-γ (10 ng/ml) for 24 h, and the protein levels of CCL5 in the supernatants were determined by ELISA. G N9 cells were pretreated with DMSO or RGFP966 (10 µM) for 12 h, then the cells were stimulated with rIFN-γ for indicating hours, and the protein levels of CCL5 in the supernatant and the protein levels of GAPDH in cells were determined via western blot. H Primary microglia were pretreated with RGFP966 for 12 h, and then were stimulated with rIFN-γ for 24 h, and the concentrations of CCL5 in the supernatant were determined by ELISA. I - J Migrated CD8 + T cells induced by conditional medium from primary microglia treated with rIFN-γ plus siRNA ( I ) or rIFN-γ plus RGFP966 ( J ) were analyzed by cell counting. K , L Migrated CD8 + T cells induced by conditional medium from N9 cells treated with rIFN-γ plus siRNA ( K ) or rIFN-γ plus RGFP966 ( L ) with or without CCL5 neutralization antibody were analyzed by cell counting. M The acetylation levels at H3K9 on the promoter of Ccl5 were determined by chromosome immunoprecipitation plus real-time PCR. M The model that HDAC3 regulates the expression of CCL5 by histone deacetylation. (* indicates p < 0.05, **indicates p < 0.01 by ANOVA or Student’s t -test)

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Counting, Neutralization, Immunoprecipitation

Journal: Cell & Bioscience

Article Title: Microglial HDAC3 inhibits the migration of CD8-positive T cell in the development of EAE by restraining the expression of CCL5

doi: 10.1186/s13578-026-01564-5

Figure Lengend Snippet: Anti-CD8 neutralizing antibody could attenuate the symptoms of Hdac3 cKO EAE mice. A Clinical score of Hdac3 cKO_EAE mice injected with anti-CD8 NAb ( n = 7) or isotype IgG ( n = 7) every other day, and WT_EAE ( n = 7) mice injected with isotype IgG were recorded every day post immunization. B , C Spinal cord slices from Hdac3 cKO_EAE mice injected with anti-CD8 NAb ( n = 6) or isotype IgG ( n = 6) every other day, and WT_EAE ( n = 6) mice injected with isotype IgG were stained with Fast-blue and eosin ( B ), and the percentage of the Fast-blue negative area ( C ) was analyzed by ImageJ. D , E Spinal cord slices from Hdac3 cKO_EAE mice injected with anti-CD8 NAb ( n = 6) or isotype IgG ( n = 6) every other day, and WT_EAE ( n = 7) mice injected with isotype IgG were stained with anti-MBP antibody and the percentage of the Fast-blue negative area was analyzed by ImageJ. F Proposed mechanism: microglial HDAC3 restrained IFN-γ-induced expression of CCL5 via deacetylation of H3K9 on the promoter of Ccl5 ; inhibition of HDAC3 resulted in upregulation of CCL5 in microglia, which promoted the migration of CD8 + T cells to the spinal cord to accelerate the development of EAE

Article Snippet: The Hdac3 flox/+ mice (Stock No: 024119) and Cx3cr1 creERT2−IRES−EYFP (also named Cx3cr1 creERT2 ) transgenic mice (Stock No: 021160) were purchased from the Jackson Laboratory (Sacramento, CA, USA) and have been used in our previous study [ ].

Techniques: Injection, Staining, Expressing, Inhibition, Migration

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: Representative images of retina flatmount immunolabeling ( A ) at 14 days post-injury and quantitative analyses ( B ) demonstrate decreased neurodegeneration indicated by the neuronal marker NeuN and a reduction in microglia/ macrophage numbers, marked by Iba-1 ( C ) in the M-HDAC3 −/− retinas ( N = 5) compared to control HDAC3 f/f retinas ( N = 7). FOV = Field of view, * p < 0.05, *** p < 0.005, **** p < 0.001.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Immunolabeling, Marker, Control

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: A , B Representative N1, P1, and N2 waveforms in the retinas of HDAC3 f/f and M-HDAC3 −/− sham and injured mice, conducted on day 7 post-ONC injury. Quantification and comparison of the ONC groups reveal improved waveform amplitudes in M-HDAC3 −/− retinas with statistical significance achieved in N2 and P1-N2 amplitudes compared to HDAC3 f/f retinas at 7 days post-ONC injury (HDAC3 f/f , N = 6 ; M-HDAC3 −/− , N = 7 )( C – F ), with no effect on the wave latencies ( G – I ). Similarly, N2 and P1-N2 amplitudes were significantly improved ( J – N ), but not their latencies ( O – Q ) at 14 days post-ONC injury (HDAC3 f/f , N = 5; M-HDAC3 −/− , N = 6). * p < 0.05, ** p < 0.01, **** p < 0.001, ns not statistically significant.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Comparison

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: M-HDAC3 −/− and HDAC3 f/f mice were subjected to ONC, and retinas were collected at days 5 (HDAC3 f/f , N = 8; M-HDAC3 −/− , N = 15), 7, and 14 days (HDAC3 f/f , N = 4; M-HDAC3 −/− , N = 5) post-injury. A Representative Z-Stack confocal images of retina flatmounts at day 5 post-ONC injury display colocalization of TUNEL + apoptotic cells (red) and Iba-1 + microglia/macrophages (green). Arrows indicate free TUNEL + apoptotic cells, while arrowheads denote Iba-1-associated apoptotic cells. B Magnification of the crosshair area from the orthogonal view and 3D rendering showing a myeloid cell wrapping its processes around an apoptotic cell. C The ratio of engulfed apoptotic cells by microglia/macrophages (Iba-1 + TUNEL + ) to free apoptotic cells was markedly increased in injured retinas of M-HDAC3 −/− mice compared to HDAC3 f/f mice, indicating improved efferocytosis on day 5 after ONC. * p < 0.05.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: TUNEL Assay

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: A Representative confocal images of optic nerve sections from M-HDAC3 −/− and HDAC3 f/f mice immunolabeled with Iba-1 (myeloid cell marker, green), CD68 (phagocytic cell marker, red), and DAPI (nuclei marker, blue) demonstrate an increase in phagocytic myeloid cells, indicated by arrows, in M-HDAC3 −/− mice compared to HDAC3 f/f on day 7 after ONC. B Representative confocal images of axonal growth and nerve fiber sprouting in the axons distal to the crush site by anterograde tracing with cholera toxin B (CTB) on day 14 post-ONC. C The quantification of the sprouting axons demonstrated significant improvement in axonal growth in M-HDAC3 −/− compared to HDAC3 f/f retinas, indicated by fluorescence intensity at distances of 200, 400, and 600 μm beyond the crush site (HDAC3 f/f , N = 5; M-HDAC3 −/− , N = 7). * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Immunolabeling, Marker, Anterograde Tracing, Fluorescence

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: A Experimental setup using myelin debris from the optic nerve labeled with Dil-red dye (top) and unlabeled debris stained with Oil Red O (ORO, bottom). B Representative images show the internalization of DiI-labeled myelin (arrows) by bone-marrow-derived macrophages derived from HDAC3 f/f and M-HDAC3 −/− mice. C Quantification of uptake of DiI-labeled myelin, expressed as mean fluorescence intensity (MFI), demonstrates significant improvement in the phagocytic activity of M-HDAC3 −/− macrophages compared to HDAC3 f/f macrophages. D ORO staining confirmed the improved uptake of myelin debris (arrows) by M-HDAC3 −/− macrophages compared to HDAC3 f/f macrophages. E Representative confocal images of Iba-1+ myeloid cells (red) and myelin basic protein (MBP, green) in optic nerve sections 7 days after ONC show a considerable increase in myelin clearance by myeloid cells in M-HDAC3 −/− optic nerves compared to HDAC3 f/f injured controls, as evidenced by increased Iba-1/MBP colocalization. N = 3 per group, **** p < 0.001.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Labeling, Staining, Derivative Assay, Fluorescence, Activity Assay

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: Macrophages from HDAC3 f/f and M-HDAC3 −/− mice were co-incubated with K-562 apoptotic cells (apop) in an in vitro efferocytosis assay. Controls included either co-incubation of macrophages with K-562 non-apoptotic cells (non-apop) or no treatment (no ttt). A – C Quantification of mRNA levels of ODC, MerTK, and the anti-inflammatory cytokine IL-10 demonstrated significant upregulation in M-HDAC3 −/− macrophages as compared to HDAC3 f/f macrophages incubated with apoptotic cells. D – F Western blotting shows significant upregulation of MerTK and ODC in M-HDAC3 −/− macrophages co-cultured with K-562 cells compared to untreated M-HDAC3 −/− macrophages, but not in the HDAC3 f/f control co-cultures. β-actin was used as a loading control. G Representative confocal images of Iba-1 + myeloid cells (green) and MerTK (red) in retinal sections 7 days after ONC show a considerable increase in MerTK expression by myeloid cells in M-HDAC3 −/− retinas compared to HDAC3 f/f controls, as evidenced by increased Iba-1/MerTK colocalization. H Similarly, MerTK expression by myeloid cells is increased in injured optic nerve sections, with arrowheads pointing to Iba-1 + MerTK + myeloid cells. GCl ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. N = 4 per group. * p < 0.05, *** p < 0.005, **** p < 0.001.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Incubation, In Vitro, Western Blot, Cell Culture, Control, Expressing

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: A Representative images illustrate the internalization of DiI-labeled myelin debris (red), derived from optic nerve axons, by CFDA-labeled macrophages (green) obtained from HDAC3 f/f and M-HDAC3 −/− mice ( N = 5 per group). These cells were pretreated with 0.8 nM of the MertK inhibitor (UNC2025) or vehicle for 1 h in vitro. B Quantification of DiI-labeled myelin debris uptake, expressed as mean fluorescence intensity (MFI), demonstrates a significant reduction in myelin uptake in the UNC2025 pretreatment groups, with UNC2025 abolishing the enhanced myelin uptake observed in vehicle-treated M-HDAC3 −/− macrophages, indicating that myeloid HDAC3 deletion promotes myelin uptake at least in part via MerTK. C Representative images of immunolabeling for neurons, marked by NeuN (green), and microglia/macrophages, marked by Iba-1 (red), of adult M-HDAC3 −/− and HDAC3 f/f mice retinas that were explanted for 24 h and treated with UNC2025 or vehicle (HDAC3 f/f , N = 3, 4; M-HDAC3 −/− , N = 5) for another 24 h. D , E Quantification of NeuN and Iba-1 shows that UNC2025 treatments had no significant effect on neuronal preservation in retinal explants of both treated groups, while it significantly increased myeloid cell number in M-HDAC3 −/− compared to flox retina explants. F , G Representative images of Iba-1 (red) labeling and quantification of optic nerve explants treated with UNC2025 show no effect of the treatment on myeloid cell count between M-HDAC3 −/− and HDAC3 f/f derived optic nerves ( N = 3 per group). * p < 0.05, **** p < 0.001; ns, not statistically significant; FOV, field of view.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Labeling, Derivative Assay, In Vitro, Fluorescence, Immunolabeling, Preserving, Cell Characterization

Journal: Cell Death Discovery

Article Title: Myeloid HDAC3 deletion protects against traumatic optic injury

doi: 10.1038/s41420-026-03030-0

Figure Lengend Snippet: A Representative retina flatmount images from microglia-specific HDAC3 KO (im-HDAC3 −/− ) and HDAC3 f/f controls immunolabeled for NeuN (neuronal marker, green) and Iba-1 (microglia/macrophages marker, red) at 14 days after ONC. B , C Quantitative analyses reveal significant neurodegeneration, indicated by a decrease in the neuronal marker NeuN and increase in Iba-1 + cell count in injured im-HDAC3 −/− ( N = 5) and HDAC3 f/f ( N = 6) mice compared to shams ( N = 3 for im-HDAC3 −/− , and N = 4 for HDAC3 f/f ). However, no differences were observed between the injured groups. D , E Representative images and Iba-1 quantification at the optic nerve injury site of im-HDAC3 −/− ( N = 5) and HDAC3 f/f ( N = 4) mice 14 days post-ONC injury show a robust presence of activated microglia and macrophages, with no differences observed between the injured groups. *** p < 0.005, **** p < 0.001; ns, not statistically significant; FOV, field of view.

Article Snippet: Sections were then labeled for Iba-1, CD68 (BioLegend, Cat. #137002), MerTk (R&D Systems, Cat. # AF591), MBP (Abcam, Cat. # ab7349), HDAC3 (Proteintech, Cat. # 10255-1-AP), and GFAP (Abcam, Cat. #13-0300).

Techniques: Immunolabeling, Marker, Cell Characterization