Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: p300‐driven H3K18ac and H3K27ac upregulates AR transcription in granulosa cells of mouse PCOS ovaries. (a) Western blotting. Ovarian tissue homogenates from Ctrl and DHEA‐treated mice were examined for p300, CBP, Gcn5, Tip60 and SRC‐3. GAPDH served as loading control. Two representative samples from each group were shown. Quantifications were shown on the right side. Data were presented as means ± SEM, n = 6. *p < 0.05 , Student's t‐test. (b) Western blotting. Ovarian tissue homogenates were assayed for H4 acetylation (H4K16ac, H4K12ac, H3K18ac, H3K27ac) and H4 (the left panel), and H3 acetylation (H4K8ac, H4K5ac, H3K14ac, H3K9ac) and H3 (the right panel). Quantifications were shown. Data were presented as means ± SEM, n = 6. *p < 0.05 , Student's t‐test. (c) Representative photomicrographs of ovarian sections from Ctrl and DHEA‐treated mice stained for p300 (red), BRD4 (green) and AR (magenta) by multiplex immunofluorescence (mIF) staining. Cell nuclei were stained with DAPI. (d) Quantifications of (c). The percentage of each fluorescent positive area relative to the DAPI area. (e) Primary granulosa cells (GCs) were treated with CTPB (5 µ m , 24 h). The cell lysates were assayed for p300 and AR. Quantifications were shown on the lower side. Data were presented as means ± SEM, n = 3, *p < 0.05 , Student's t ‐test. (f) Primary granulosa cells (GCs) were treated with DHEA (25 µ m , 48 h) followed by C646 (10 µ m , 24 h) or A‐485 (1 µ m , 24 h) treatment, and then cell lysates were assayed for p300, AR, H3K18ac, H3K27ac, H4K16ac, H4K12ac, H3, H4, and GAPDH. (g) Quantification of (e). Data were presented as means ± SEM, n = 4, *P < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test. (h) Luciferase assay. HEK293T cells were transfected with an AR promoter‐luciferase reporter AR‐luc plus a renilla luciferase reporter, and then treated with DHEA (25 µ m , 48 h) with or without C646 (10 µ m , 24 h). Cell lysates were assayed for luciferase activities. The relative luciferase activities of fold changes were presented. Data were presented as means ± SEM, n = 4, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Primary antibodies used included: p300 (ab275378, Abcam), BRD4 (83375S, CST, USA), AR (ab52615, Abcam), Histone3 lysine site 9 acetylated antibody (H3K9ac, ab4441, abcam), H3K14ac (HY‐ P80167 , MCE), H3K18ac (13998T, CST), H3K27ac (8173T, CST), H4K5ac (HY‐ P80182 , MCE), H4K8ac (HY‐ P80180 , MCE), H4K12ac (ab46983, abcam), H4K16ac (HY‐ P80179 , MCE), Histone3 (H3, 17168‐1‐AP, Proteintech), Histone4 (H4, 16047‐1‐AP, Proteintech), α‐SMA (BS70000, Bioworld), CBP (AG1691, Beyotime), Gcn5 ( PA002657 , CUSBIO), Tip60 ( PA006616 , CUSBIO), SRC‐3 (ER65603, HUABO), Col1α (BS1530, Bioworld), SP1 (A19649, ABclonal), and GAPDH (FD0063, FDbio).

Techniques: Western Blot, Control, Staining, Multiplex Assay, Immunofluorescence, Luciferase, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Histone Acetylation Landscape and the Role of HAT1 in Regulating Corneal Epithelial Wound Healing

doi: 10.1167/iovs.67.2.45

Figure Lengend Snippet: Histone acetylation mapping and elevated levels of HAT1 and H4K5Ac during corneal epithelial wound healing process. ( A ) Representative Western blots showing the screening results for acetylation modifications at various histone lysine residues (H3K9, H3K18, H3K27, H3K36, H4K5, H4K8, and H4K12) in corneal epithelial samples from control (CON) and wound healing (WH) groups. Notably, acetylation at H4K5 (H4K5Ac) and H3K27 (H3K27Ac) were significantly increased in WH samples compared with controls, whereas other sites exhibited no apparent changes. ( B ) Expanded Western blot validation for H4K5Ac levels in the CON and WH groups. ( C ) Representative Western blot analysis of HAT1 protein expression in CON and WH samples. ( D , E ) Densitometric quantification showing an approximately three-fold increase in H4K5Ac levels ( n = 7/group; P < 0.01) normalized to total histone H4 ( D ), and HAT1 levels ( n = 10/group; P < 0.001) normalized to β-actin ( E ) in WH samples relative to controls. Data are presented as mean ± SEM.

Article Snippet: The sheared chromatin samples were incubated with H4K5Ac antibody (CST) or IgG isotype control overnight at 4°C.

Techniques: Western Blot, Control, Biomarker Discovery, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Histone Acetylation Landscape and the Role of HAT1 in Regulating Corneal Epithelial Wound Healing

doi: 10.1167/iovs.67.2.45

Figure Lengend Snippet: Generation and validation of corneal epithelium-specific Hat1 cKO mice. ( A ) Schematic illustration of the Cre-loxP strategy used to generate Hat1 cKO mice. Krt12 promoter-driven Cre recombinase mice (Krt12-Cre) were crossed with mice carrying floxed alleles of the Hat1 gene ( Hat1 flox/flox ) to achieve corneal epithelial cell-specific deletion of Hat1 . ( B ) PCR-based genotyping results from mouse tail biopsies, confirming the presence of Cre (280 bp) and the floxed Hat1 allele (339 bp). ( C ) RT-qPCR analysis showing a significant reduction (approximately 95%) in Hat1 mRNA levels in corneal epithelial tissues from Hat1 cKO mice compared with Hat1 flox/flox controls (n = 6/group; P < 0.001). ( D ) Western blot analysis and densitometric quantification demonstrating marked decreases in HAT1 protein expression and H4K5Ac level in corneal epithelium from Hat1 cKO mice relative to controls ( n = 6/group; P < 0.001). ( E ) Representative immunofluorescence images of corneal sections showing prominent HAT1 expression ( green ) in basal epithelial cells of control mice, while signal was absent in Hat1 cKO mice. Cell nuclei are stained with DAPI ( blue ). Scale bar , 50 µm. Data are presented as mean ± SEM.

Article Snippet: The sheared chromatin samples were incubated with H4K5Ac antibody (CST) or IgG isotype control overnight at 4°C.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Control, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Histone Acetylation Landscape and the Role of HAT1 in Regulating Corneal Epithelial Wound Healing

doi: 10.1167/iovs.67.2.45

Figure Lengend Snippet: HAT1 regulates corneal epithelial cell proliferation via modulating the G1-to-S phase transition. ( A ) Western blot analysis showed significant knockdown of HAT1 protein and corresponding reduction of H4K5Ac levels after transfection with HAT1-specific siRNAs (siHAT1-I and siHAT1-II) in HCECs, while acetylation at other histone sites remained unchanged. ( Right ) Densitometric quantification of HAT1 and H4K5Ac levels in NC and HAT1 siRNA transfected groups ( n = 4/group; P < 0.001). ( B ) MTS assays demonstrated reduced proliferation of HCECs upon HAT1 knockdown compared with NC cells at the indicated time points. The y axis represents the optical density (OD) at 490 nm, which is proportional to cell number ( n = 6/group; P < 0.01). ( C , D ) EdU incorporation assays showed significantly fewer proliferative (EdU-positive, green ) cells after HAT1 knockdown; nuclei were counterstained with DAPI ( blue ). Quantification was performed from four random fields of view (FOV) per experiment, and the experiment was repeated three times. The y axis represents the percentage of EdU+ cells among DAPI+ cells ( n = 3/group; P < 0.01). Scale bar , 50 µm. ( E , F ) Flow cytometry-based cell-cycle analyses revealed a significant increase in G1-phase cell population after HAT1 knockdown ( n = 3/group; P < 0.01). ( G ) Western blot analysis confirmed successful overexpression of HAT1 and increased levels of H4K5Ac in HCECs infected with Lv-HAT1 lentivirus. ( Right ) Densitometric quantification of H4K5Ac levels ( n = 3/group; P < 0.01). ( H ) MTS assays indicated enhanced cell proliferation after HAT1 overexpression compared with the control group ( n = 6/group; P < 0.01). ( I , J ) Flow cytometry analysis demonstrated a decreased G1-phase and increased S-phase cell population in Lv-HAT1 infected cells compared with controls ( n = 3/group; P < 0.05). Data are presented as mean ± SEM.

Article Snippet: The sheared chromatin samples were incubated with H4K5Ac antibody (CST) or IgG isotype control overnight at 4°C.

Techniques: Sublimation, Western Blot, Knockdown, Transfection, Flow Cytometry, Over Expression, Infection, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Histone Acetylation Landscape and the Role of HAT1 in Regulating Corneal Epithelial Wound Healing

doi: 10.1167/iovs.67.2.45

Figure Lengend Snippet: H4K5Ac is enriched at the CCND1 and CDK6 promoters and is dependent on HAT1. ( A ) ChIP-qPCR assays showed that H4K5Ac was significantly enriched at the promoter regions of CCND1 and CDK6 in HCECs, with IgG serving as a NC ( n = 3/group; P < 0.01). ( B ) ChIP-qPCR quantifying H4K5ac enrichment at the CCND1 and CDK6 promoter regions in siHAT1- vs. NC-transfected HCECs. Data are shown as percent input (% input) after subtraction of IgG background ( n = 3/group; P < 0.01). GAPDH served as a nonresponsive NC locus, and YWHAZ was included as an additional control locus. Data are presented as mean ± SEM.

Article Snippet: The sheared chromatin samples were incubated with H4K5Ac antibody (CST) or IgG isotype control overnight at 4°C.

Techniques: ChIP-qPCR, Transfection, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Histone Acetylation Landscape and the Role of HAT1 in Regulating Corneal Epithelial Wound Healing

doi: 10.1167/iovs.67.2.45

Figure Lengend Snippet: Schematic illustration of the epigenetic mechanism by which HAT1 regulates corneal epithelial wound healing. Corneal injury significantly upregulates nuclear HAT1 expression, enhancing H4K5Ac at the CCND1 and CDK6 promoters, which is associated with increased transcription. The resulting CCND1 and CDK6 proteins stimulate corneal epithelial cell proliferation, ultimately facilitating wound closure and reepithelialization.

Article Snippet: The sheared chromatin samples were incubated with H4K5Ac antibody (CST) or IgG isotype control overnight at 4°C.

Techniques: Expressing

Journal: bioRxiv

Article Title: SLAMF1-peptide mediated epigenetic priming reprograms innate immune responses in sepsis

doi: 10.64898/2025.12.29.696918

Figure Lengend Snippet: (A) Table shows results of a mass spectrometry–based screen identifying lysine deacetylases co-precipitating with P7-Pen compared with control peptide (Pen), based on three independent experiments. (B) Western blot analysis of ABHD14B and TRAM (known P7-interacting protein, positive control) in pulldown samples from monocyte lysates using biotinylated Pen or P7-Pen peptides immobilized on streptavidin beads, with whole-cell lysates included as input controls (7.5% of total protein); total protein staining by SimplyBlue is shown as a loading control for peptides. (C) The efficiency of ABHD14B silencing in primary human macrophages was assessed by RT–qPCR following transfection with four independent A BHD14B -targeting siRNAs, with results expressed as mean relative fold change ± SEM from three independent experiments. (D) Representative immunoblots illustrate the effects of ABHD14B knockdown on histone acetylation (H3K9ac and H4K5ac) and STAT1 phosphorylation, with β-tubulin used as a loading control. (E–G) Functional validation of ABHD14B silencing was performed in macrophages from six healthy donors, demonstrating efficient knockdown at the protein level ( E ), reduced transcript abundance by qPCR ( F ), and altered LPS-induced secretion of IL-6, MCP-1, IP-10, and TNF ( G ) as measured by multiplex cytokine analysis. Data are presented as mean relative fold change ± SEM, and statistical significance was determined using nonparametric one-way repeated-measures ANOVA ( p < 0.05).

Article Snippet: The following primary antibodies were used: mouse β-tubulin (D3U1W, #86298), STAT1 (9H2, #9176), rabbit histone H3 (D1H2, #4499), H3K9ac (acetyl-histone H3 Lys9, C5B11, #9649), H3K27ac (acetyl-histone H3 Lys27, D5E4, #8173), H3K56ac (acetyl-histone H3 Lys56, #4243), H4K5ac (acetyl-histone H4 Lys5, D12B3, #8647), H4K12ac (acetyl-histone H4 Lys12, D2W60, #13944), phospho-STAT1 (Tyr701) (58D6, #9167) from Cell Signaling Technology (Danvers, MA, USA); mouse CD8a (1G2B10, #66868-1-Ig), rabbit CD3 (#17617-1-AP), CD14 (#17000-1-AP) were from Proteintech (); acetylated lysine mouse monoclonal Abs (MA1-2021) were from Invitrogen (Themo Fisher Scientific,); rabbit anti-ABHD14B (in-house, Pune, India) [refs].

Techniques: Mass Spectrometry, Control, Western Blot, Positive Control, Staining, Quantitative RT-PCR, Transfection, Knockdown, Phospho-proteomics, Functional Assay, Biomarker Discovery, Multiplex Assay