hdac4  (Proteintech)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: NAT10 stimulates HDAC4 expression via ac4C modification. A Volcano plot of differentially expressed ac4C acetylation peaks ( P < 0.05) identified by acRIP-seq in NAT10 knockdown cells. B Distribution of ac4C peaks across mRNA regions in breast cancer cells. C Sequence logo of the enriched motif within ac4C peaks identified by HOMER. D Volcano plot of differentially expressed mRNAs ( P < 0.05) identified by RNA-seq in NAT10 knockdown cells. E Integrative analysis of acRIP-seq and RNA-seq data to identify potential downstream targets of NAT10. F , G Relative HDAC4 mRNA expression measured by qRT-PCR after NAT10 knockdown. H , I acRIP-qPCR analysis of ac4C modification ( H ) and RIP-qPCR analysis of NAT10 binding on HDAC4 mRNA ( I ) after NAT10 knockdown. J Genome browser view of ac4C peaks on HDAC4 mRNA from acRIP-seq. K Relative HDAC4 protein levels measured by Western blot after NAT10 knockdown. L qRT-PCR analysis of HDAC4 mRNA stability after actinomycin D treatment in NAT10 knockdown cells. M Luciferase activity of the reporter constructs containing the wild-type or ac4C site mutated sequence in NAT10 knockdown cells. N , O HDAC4 expression assessed by qRT-PCR ( N ) and Western blot ( O ) after transfection with oeNAT10 or NAT10 G641E mutant plasmid. P qRT-PCR analysis of HDAC4 mRNA stability after actinomycin D treatment in cells transfected with oeNAT10 or NAT10 G641E . Q , R acRIP-qPCR analysis of ac4C modification ( Q ) and RIP-qPCR analysis of NAT10 binding on HDAC4 mRNA ( R ) after transfection with oeNAT10 or NAT10 G641E . S Luciferase activity of the reporter constructs containing the wild-type or ac4C site mutated sequence after transfection with oeNAT10 or NAT10 G641E . All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The HDAC4 inhibitor LMK-235 (MedChemExpress, #HY18998, China) was administered intraperitoneally at doses of 5 mg/kg or 10 mg/kg body weight every three days.

Techniques: Expressing, Modification, Knockdown, Sequencing, RNA Sequencing, Quantitative RT-PCR, Binding Assay, Western Blot, Luciferase, Activity Assay, Construct, Transfection, Mutagenesis, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: HDAC4 acts as a deacetylase to regulate NAT10 stability. A Representative IHC images and quantification of NAT10 and HDAC4 expression in breast cancer tissues ( n = 220; scale bar, 200 µm). B , C Correlation between NAT10 and HDAC4 expression levels in breast cancer tissues, as analyzed by IHC ( B ) and in the TCGA-BRCA RNA-seq dataset ( C) . D , E Relative NAT10 expression was detected by Western blot ( D ) and qRT-PCR ( E ) after HDAC4 knockdown or overexpression. F Western blot analysis of NAT10 protein stability after cycloheximide treatment in cells with HDAC4 knockdown or overexpression. G Molecular docking model of the NAT10 (blue) and HDAC4 (yellow) interaction, with an enlarged view highlighting predicted hydrogen bonds. H Co-IP followed by Western blot analysis was used to detect the interaction between endogenous HDAC4 and NAT10. I Co-IP followed by Western blot analysis assessed NAT10 acetylation levels after HDAC4 knockdown or overexpression. J Prediction of potential deacetylation sites on NAT10 using MusiteDeep. K The predicted acetylation sites on NAT10 were individually mutated (K→R), and the effects of HDAC4 on the acetylation levels of the six NAT10 mutants were examined in 293T cells. L Detection of NAT10 acetylation in cells expressing NAT10 K354R following transfection with vector or HDAC4. M Detection of NAT10 acetylation in cells transfected with wild-type or catalytically inactive (D840N) HDAC4. N Western blot analysis of NAT10 protein stability following cycloheximide treatment in cells transfected with siNC, siHDAC4, or siHDAC4 plus NAT10 K354R . All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The HDAC4 inhibitor LMK-235 (MedChemExpress, #HY18998, China) was administered intraperitoneally at doses of 5 mg/kg or 10 mg/kg body weight every three days.

Techniques: Histone Deacetylase Assay, Expressing, RNA Sequencing, Western Blot, Quantitative RT-PCR, Knockdown, Over Expression, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: The proliferative function of NAT10 in breast cancer depends on HDAC4. A CCK-8 assays of cells transfected with vector, oeNAT10, oeNAT10 plus siHDAC4-1, or oeNAT10 plus siHDAC4-2. B Flow cytometry analysis of apoptotic rates (LR + UR) in cells transfected with the indicated constructs. C Flow cytometry analysis of cell cycle distribution in cells transfected with the indicated constructs. D Representative images of tumors in mice treated with vector, oeNAT10, oeNAT10 plus LMK235 (5 mg/kg), or oeNAT10 plus LMK235 (10 mg/kg) ( n = 6). E Tumor growth curves and tumor weight analysis in tumors from different groups ( n = 6). F Representative IHC images of NAT10, HDAC4, and Ki67 in tumor tissues from different treatment groups, with quantification of staining intensity ( n = 6; scale bars, 50 μm). G Representative TUNEL staining of tumor tissues from different treatment groups, with quantification of staining intensity ( n = 6; scale bars, 100 px). H-J CCK-8 assays ( H ) and flow cytometry analyses of apoptotic rates ( I ) and cell cycle distribution ( J ) in cells transfected with vector, oeNAT10, oeNAT10 plus LMK235 (1 µM), or oeNAT10 plus LMK235 (2 µM). All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The HDAC4 inhibitor LMK-235 (MedChemExpress, #HY18998, China) was administered intraperitoneally at doses of 5 mg/kg or 10 mg/kg body weight every three days.

Techniques: CCK-8 Assay, Transfection, Plasmid Preparation, Flow Cytometry, Construct, Staining, TUNEL Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: NAT10 promotes PD-L1 expression through HDAC4. A Relative PD-L1 expression assessed by Western blot and qRT–PCR following NAT10 knockdown or overexpression. B IF staining of PD-L1 in cells with NAT10 knockdown or overexpression (scale bars, 5 μm). C Representative IHC images of PD-L1 in tumor tissues from shNC or shNAT10 groups and vector or oeNAT10 groups, with quantification of staining intensity ( n = 6; scale bars, 50 μm). D-F Relative PD-L1 expression analyzed by Western blot ( D ), qRT-PCR ( D ), and IF ( E-F ) in cells transfected with vector, oeNAT10, oeNAT10 plus siHDAC4-1, or oeNAT10 plus siHDAC4-2. G Representative IHC images of PD-L1 in tumor tissues from vector, oeNAT10, oeNAT10 plus LMK235 (5 mg/kg), or oeNAT10 plus LMK235 (10 mg/kg), with quantification of staining intensity ( n = 6; scale bars, 50 μm) All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The HDAC4 inhibitor LMK-235 (MedChemExpress, #HY18998, China) was administered intraperitoneally at doses of 5 mg/kg or 10 mg/kg body weight every three days.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Knockdown, Over Expression, Staining, Plasmid Preparation, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: NAT10 promotes PD-L1 expression through the HDAC4–NF-κB pathway. A GSEA of RNA-seq (siHDAC4 vs. siNC) data showed enrichment of the NF-κB signaling pathway (NES, normalized enrichment score; P value by permutation test). B Correlation analysis of p65, NAT10, HDAC4, and PD-L1 mRNA expression in the TCGA RNA-seq dataset. C Relative PD-L1 expression assessed by qRT-PCR following HDAC4 knockdown or overexpression. D Western blot analysis of PD-L1 and NF-κB pathway proteins upon HDAC4 knockdown or overexpression. E Predicted p65 binding sites in the PD-L1 promoter identified using the JASPAR database. F ChIP-qPCR analysis of p65 enrichment at the PD-L1 promoter after HDAC4 knockdown or overexpression. G Western blot analysis of NF-κB pathway proteins in cells transfected with vector, oeNAT10, oeNAT10 plus siHDAC4-1, or oeNAT10 plus siHDAC4-2. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The HDAC4 inhibitor LMK-235 (MedChemExpress, #HY18998, China) was administered intraperitoneally at doses of 5 mg/kg or 10 mg/kg body weight every three days.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Binding Assay, ChIP-qPCR, Transfection, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: Mode pattern of the NAT10/HDAC4/NF-κB regulatory network in breast cancer. NAT10 mediated ac4C modification stabilizes HDAC4 mRNA, while HDAC4 stabilizes NAT10 protein, forming a reciprocal regulatory loop. HDAC4 activates NF-κB signaling, leading to PD-L1 upregulation and immune evasion. Inhibition of the NAT10/HDAC4/NF-κB axis reduces PD-L1 expression and restores antitumor immunity in breast cancer

Article Snippet: The HDAC4 inhibitor LMK-235 (MedChemExpress, #HY18998, China) was administered intraperitoneally at doses of 5 mg/kg or 10 mg/kg body weight every three days.

Techniques: Modification, Inhibition, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A, VS-6063 reduced levels of phosphorylated HDAC4 and HDAC5. n=4 biological replicates in each group. B, Silencing FAK also reduced the phosphorylation of HDAC4 and HDAC5 in HASMCs grown in common medium or osteogenic medium. n=4 biological replicates in each group. C, FAK inhibition using VS6063 decreased cytosolic localization and increased nuclear localization of HDAC4 and HDAC5 in osteogenic media as shown by immunofluorescence images (60x). 30 cells in each group were used for analysis.

Article Snippet: Recombinant adenoviruses expressing human HDAC4 or HDAC5 were obtained from Vector Biolabs (HDAC4: #1435; HDAC5: #210890).

Techniques: Inhibition, Phospho-proteomics, Immunofluorescence

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: Leptomycin B (LEP) inhibited nuclear export of HDAC4 and HDAC5 in A , normal media and B , in osteogenic media as shown by immunofluorescence. 12–46 cells in each group were used for analysis. C, Leptomycin B reduced phosphorylated HDAC4 and HDAC5 and inhibited the expression of RUNX2 and ALPL in osteogenic media. n=4 biological replicates in each group.

Article Snippet: Recombinant adenoviruses expressing human HDAC4 or HDAC5 were obtained from Vector Biolabs (HDAC4: #1435; HDAC5: #210890).

Techniques: Inhibition, Immunofluorescence, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A , Increased expression of HDAC4/5 was achieved by adenovirus transduction (Ad.HDAC4 or Ad.HDAC5). B, Ad.HDAC4 or Ad.HDAC5 resulted in enhanced calcification that was inhibited with the treatment of leptomycin B (5nM) n=3 biological replicates in each group (with 2 representative replicates shown). Quantitative calcium assay in cells treated with C, Ad.HDAC4 and D , Ad.HDAC5 in the presence or absence of leptomycin demonstrated reduced calcification with leptomycin treatment. n=3 biological replicates in each group. E and F, Cytosolic and nuclear localization of HDAC4 and HDAC5 in AdHDAC4 and AdHDAC5 treated cells in the presence and absence of leptomycin (a nuclear export inhibitor, 10 nM for 3 hours) in osteogenic media. 12–13 cells in each group were used for analysis.

Article Snippet: Recombinant adenoviruses expressing human HDAC4 or HDAC5 were obtained from Vector Biolabs (HDAC4: #1435; HDAC5: #210890).

Techniques: Over Expression, Expressing, Transduction, Calcium Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A, Treatment of HASMCs with siHDAC4 resulted in >70% and >65% knockdown of HDAC4 mRNA level in common or osteogenic medium, respectively. n=6 biological replicates in each group. B, Protein levels of RUNX2 and ALPL were increased with osteogenic medium. However, siHDAC4 decreased the levels of RUNX2 and ALPL induced by osteogenic medium. n=4 biological replicates in each group. C, Treatment of HASMCs with siHDAC5 resulted in >55% and >50% knockdown of HDAC5 mRNA level in normal or osteogenic medium, respectively, and decreased HDAC5 protein levels in siHDAC5-treated cells. n=6 biological replicates in each mRNA group. D, siHDAC5 decreased the protein levels of RUNX2 and ALPL induced by osteogenic medium. n=4 biological replicates in each group. E, Treatment with siHDAC4, siHDAC5, or the combination inhibited calcification of HASMCs grown in osteogenic medium for 14 days, as evidenced by Alizarin Red staining. n=3 biological replicates in each group (with 2 representative replicates shown). F, Treatment of HASMCs with LMK-235 (a pharmacologic inhibitor of HDAC4 and HDAC5) inhibited calcification induced by osteogenic medium in a dose-dependent manner. n=2 biological replicates in each group G, LMK-235 reduced the migration of VSMCs induced by osteogenic medium. The experiments in figure 1H and 7G were performed at the same time. n=6 biological replicates in each group.

Article Snippet: Recombinant adenoviruses expressing human HDAC4 or HDAC5 were obtained from Vector Biolabs (HDAC4: #1435; HDAC5: #210890).

Techniques: Inhibition, Knockdown, Staining, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A and B, Increased HDAC4 or HDAC5 expression by adenovirus resulted in augmented calcification of HASMCs that was inhibited by treatment with the FAK inhibitor VS-6063 (1μM). n=3 biological replicates in each group (with 2 representative replicates shown). C and D, Reduction of FAK expression with siFAK significantly attenuated the calcification of HASMCs induced by Ad.HDAC4 or Ad.HDAC5 in osteogenic medium or induced by osteogenic medium alone. n=3 biological replicates in each group (with 2 representative replicates shown). E and F, Osteogenic medium induced calcification of mouse aortas and human carotid arteries after culturing for 21 days. However, the calcification induced by osteogenic medium was inhibited by treatment with VS-6063 (2μM or 4μM). n=4 biological replicates in each group.

Article Snippet: Recombinant adenoviruses expressing human HDAC4 or HDAC5 were obtained from Vector Biolabs (HDAC4: #1435; HDAC5: #210890).

Techniques: Inhibition, Over Expression, Cell Culture, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: Localization of PTK2 , HDAC5 , and HDAC4 gene expression in modulated SMC subtypes using an integrated human atherosclerosis reference. Uniform Manifold Approximation and Projection (UMAP) embeddings from an integrated human atherosclerosis single-cell RNA-seq reference dataset (see Methods ), highlighting ( A ) PTK2 , ( B ) HDAC5 , and ( C ) HDAC4 normalized gene expression. Individual sequencing libraries across four studies were harmonized after QC and batch correction with reciprocal PCA (rPCA). A broad SMC cluster was annotated using transfer learning with cell labels from the Tabula Sapiens vasculature subset. SMC subtypes were further annotated by extracting gene modules from a scRNA meta-analysis of murine SMCs (contractile SMC, transitional SMC, fibromyocyte, and fibrochondrocyte) and calculating their enrichment in cells within the main SMC cluster. PTK2 and HDAC5 expression were enriched in transitional SMCs and fibromyocytes.

Article Snippet: Recombinant adenoviruses expressing human HDAC4 or HDAC5 were obtained from Vector Biolabs (HDAC4: #1435; HDAC5: #210890).

Techniques: Gene Expression, RNA Sequencing, Sequencing, Expressing

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: Histone H4K5la activates transcription of HDAC4 in FSH-treated GCs. (A) Elaborate examination of H4K5la binding at various genomic locations within target genes. (B) Strategy for identifying specific downstream targets of H4K5la based on CUT&Tag data. (C) Computational biology suggests that HDAC4 is a possible H4K5la effector. (D) Representative Integrative Genomics Viewer (IGV) tracks showing enriched H4K5la modifications at the HDAC4 promoter in mGCs using CUT&Tag analysis. (E) Assessment of HDAC4 mRNA abundance via qRT-PCR in mGCs and KGN cells exposed to 15 mM oxamate for 2 h, and then subjected to 5 IU of FSH for 12 h. (F) qRT-PCR was performed to assess HDAC4 mRNA expression in mGCs and KGN cells after 12-h transfection with LDHA- and LDHB-targeting siRNAs, followed by a 12-h exposure to 5 IU of FSH. (G) ChIP-qPCR revealed H4K5la enrichment at the HDAC4 promoter in KGN cells pretreated with 15 mM oxamate for 2 h before FSH stimulation (5 IU, 12 h). (H) ChIP-qPCR demonstrated H4K5la binding to the HDAC4 promoter in KGN cells transfected with LDHA and LDHB siRNAs for 12 h, followed by 5 IU of FSH treatment for an additional 12 h. (I) ChIP-qPCR analysis indicated H4K5la occupancy at the HDAC4 promoter in KGN cells incubated with 10 μM C646 for 2 h prior to 12-h FSH (5 IU) exposure. (J) Western blotting was used to evaluate HDAC4 protein levels in GCs treated with 15 mM oxamate for 2 h, followed by 5 IU of FSH for 12 h. (K) HDAC4 protein expression in (J) was quantified by densitometry and normalized to TUBA1A as a loading control. (L) Western blot detection of HDAC4 expression in mGCs and KGN cells after 12 h siRNA knockdown of LDHA/LDHB and subsequent 12-h 5-IU FSH exposure. (M) The protein levels of HDAC4 in (L) were quantitatively analyzed with normalization to TUBA1A. (N) Immunoblotting analysis assessing HDAC4 protein abundance in mGCs and KGN cells after 2-h exposure to 10 μM C646, and then 12 h of 5-IU FSH administration. (O) The protein levels of HDAC4 in (N) were quantitatively analyzed with normalization to TUBA1A. Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, ChIP-qPCR, Incubation, Western Blot, Control, Knockdown, Quantitative Proteomics

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: Histone lactylation promotes mitochondrial biogenesis in GCs via HDAC4. (A) Western blot analysis of HDAC4 protein levels in KGN cells cultured with different concentrations of LMK-235 for 12 h. (B) The protein levels of HDAC4 in (A) were quantitatively analyzed with normalization to TUBA1A. (C) qRT-PCR quantification of mitochondrial DNA ( MT-CO2 and D-Loop ) in KGN cells after 2-h exposure to 15 mM LMK-235, and then 12 h with 5 IU of FSH. β-Actin served as the loading control for data normalization. (D) TOM20 protein levels in mGCs and KGN cells were analyzed by Western blot under the following treatment: pretreatment with 15 mM LMK-235 for 2 h, followed by stimulation with 5 IU of FSH for 12 h. (E) TOM20 protein levels in (D) were quantified and normalized to TUBA1A. (F) mGCs and KGN were pretreated with 15 mM LMK-235 for 2 h and then exposed to 5 IU of FSH for 12 h. Mitochondrial labeling was performed using MitoTracker Green (Mito Green) (green), and samples were visualized via confocal microscopy. Scale bar, 5 μm. (G) Quantitative analysis of MitoTracker Green fluorescence intensity from (F). (H) Western blot examination of HDAC4 protein abundance in KGN cells treated with HDAC4 siRNA or control siRNA over a 24-h period. (I) The protein levels of HDAC4 in (H) were quantitatively analyzed with normalization to TUBA1A. (J) qRT-PCR evaluation of mitochondrial DNA copy number in KGN cells post-HDAC4 siRNA transfection for 12 h, and then treated with 5 IU of FSH for another 12 h. β-Actin served as the loading control for data normalization. (K) Western blot assessment of TOM20 expression in KGN cells after HDAC4 siRNA transfection (12 h) and subsequent FSH treatment (5 IU, 12 h). (L) The protein levels of TOM20 in (K) were quantitatively analyzed with normalization to TUBA1A. (M) KGN cells received 12 h of HDAC4-specific siRNA transfection and then underwent 12 h of 5-IU FSH treatment. Mitochondria were visualized using MitoTracker Green and imaged by laser confocal scanning microscopy. Scale bar, 5 μm. (N) Quantitative analysis of MitoTracker Green fluorescence intensity from (M). (O) KGN cells received HDAC4-targeting siRNAs for 12 h and then 5 IU of FSH for 12 h. Subsequently, OCR was quantified. (P) KGN cells underwent HDAC4 siRNA transfection for 12 h and then received 5 IU of FSH for another 12 h. JC-1 staining measured mitochondrial membrane potential. (Q) The membrane potential levels in (P) were analyzed. Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Western Blot, Cell Culture, Quantitative RT-PCR, Control, Labeling, Confocal Microscopy, Fluorescence, Quantitative Proteomics, Transfection, Expressing, Confocal Laser Scanning Microscopy, Staining, Membrane

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: FSH-induced deacetylation of PGC-1α by HDAC4 promotes mitochondrial biogenesis in GCs. (A) Conservation analysis of the PGC-1α K329/330 acetylation site across different species. (B) Analysis by co-IP reveals the engagement of PGC-1α with acetylated lysines, as assessed post-10 μM C646 administration for 2 h and then subjected to 5 IU of FSH exposure for 12 h in KGN cells. (C) Assessment of PGC-1α acetylation levels quantitatively in (B). The level of acetylation was calculated as the ratio of the pan-acetylated-lysine signal to the total PGC-1α signal following co-IP. (D) IP analysis identified the association of PGC-1α with pan-acetylated lysines post-HDAC4 silencing for 12 h, subsequent to 12 h of 5-IU FSH treatment in KGN cells. (E) Quantitative analysis of the acetylation modification level of PGC-1α in (D). The level of acetylation was calculated as the ratio of the pan-acetylated-lysine signal to the total PGC-1α signal following co-IP. (F) IP technique to identify the association of PGC-1α with all acetylated lysines in PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h, and then treated with 5 IU of FSH for 12 h. (G) Quantitative analysis of the acetylation modification level of PGC-1α in (F). The level of acetylation was calculated as the ratio of the pan-acetylated-lysine signal to the total Flag-PGC-1α signal following co-IP. (H) qRT-PCR analysis of mitochondrial DNA copy number ( MT-CO2 and D-Loop ) in PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h. Following transfection, the cells were stimulated with 5 IU of FSH for an additional 12-h period before analysis. β-Actin served as the loading control for data normalization. (I) Western blot analysis of TOM20 protein levels in KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α, or K329/330Q PGC-1α for 12 h, followed by treatment of 5 IU of FSH. (J) The protein levels of TOM20 in (I) were quantitatively analyzed with normalization to TUBA1A. (K) PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h, followed by 5 IU of FSH for an additional 12 h. MitoTracker Green (green) labeled mitochondria, visualized via laser confocal microscopy. Scale bar, 5 μm. (L) Quantitative analysis of MitoTracker Green fluorescence intensity from (K). (M) PGC-1α knockdown KGN cells overexpressing Flag-tagged WT PGC-1α, K329/330R PGC-1α (acetylation-resistant), or K329/330Q PGC-1α (acetylation-mimic) for 12 h, followed by 5 IU of FSH for an additional 12 h. The OCRs were then measured. Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Co-Immunoprecipitation Assay, Modification, Knockdown, Quantitative RT-PCR, Transfection, Control, Western Blot, Labeling, Confocal Microscopy, Fluorescence

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: Deacetylation of PGC-1α enhances its interaction with NRF1/2. (A) Analysis of the interaction between PGC-1α and NRF1/2 by IP in KGN cells. Cells were first treated with 15 mM oxamate for 2 h, followed by stimulation with 5 IU of FSH for 12 h. (B) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (A). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (C) Co-IP assays examining PGC-1α binding to NRF1/2 within KGN cells: samples pretreated with 10 μM C646 (2 h) and then stimulated with FSH (12 h). (D) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (C). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (E) Co-IP assay assessing PGC-1α and NRF1/2 binding in KGN cells post-HDAC4 knockdown (12 h) and FSH exposure (5 IU, 12 h). (F) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (E). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (G) Co-IP analysis of PGC-1α/NRF1/2 binding dynamics in KGN cells expressing Flag-tagged WT, K329/330R (acetylation-resistant), or K329/330Q (acetylation-mimic) PGC-1α, treated with or without 5 IU of FSH for 12 h. (H) Quantitative measurement of the binding affinity between PGC-1α and proteins NRF1/NRF2 in (G). The affinity is presented as the level of NRF1 or NRF2 co-immunoprecipitated with PGC-1α, normalized to the total PGC-1α protein level. (I) KGN cells were first transfected with PGC-1α siRNA for 12 h, followed by overexpression of Flag-tagged constructs: WT PGC-1α, acetylation-resistant K329/330R PGC-1α, or acetylation-mimetic K329/330Q PGC-1α for 12 h, and then treated with 5 IU of FSH for an additional 12 h. ChIP analysis of the binding of Flag-tagged PGC-1α to the promoters of Tfb1m , Tfb2m , and Tfam. (J) KGN cells overexpressing Flag-tagged WT PGC-1α plasmid for 12 h were sequentially treated with 15 μM LMK-235 (2 h) followed by 5 IU of FSH (12 h). Subcellular fractionation was then performed to obtain cytosolic and nuclear extracts, which were subjected to immunoblot analysis using antibodies against Flag (transgene expression), TUBA1A (cytosolic marker), and histone H4 (nuclear marker). (K) PGC-1α levels in the nuclear and cytoplasmic fractions were quantified in (J). H4 and TUBA1A were used as internal controls for normalizing the nuclear and cytoplasmic proteins, respectively. (L) Immunoblot analysis was performed to examine Flag-tagged WT PGC-1α expression and subcellular localization in HDAC4-knockdown KGN cells. After 12-h Flag-PGC-1α induction, cells received 5 IU of FSH for another 12 h, and cytosolic and nuclear fractions were probed for Flag, TUBA1A (cytosolic marker), and histone H3 (nuclear marker). (M) Quantitatively measure the subcellular distribution of PGC-1α between the nucleus and cytoplasm in (L). H4 and TUBA1A were used as internal controls for normalizing the nuclear and cytoplasmic proteins, respectively. (N) KGN cells were first transfected with PGC-1α siRNA for 12 h, followed by overexpression of Flag-tagged constructs: WT PGC-1α, acetylation-resistant K329/330R PGC-1α, or acetylation-mimetic K329/330Q PGC-1α for 12 h, and then treated with 5 IU of FSH for an additional 12 h. (O) Quantitatively measure the subcellular distribution of PGC-1α between the nucleus and cytoplasm in (N). H4 and TUBA1A were used as internal controls for normalizing the nuclear and cytoplasmic proteins, respectively. (P) Immunofluorescence analysis of PGC-1α subcellular localization in KGN cells transfected with Flag-tagged WT, K329/330R, or K329/330Q PGC-1α for 12 h, followed by treatment 5 IU of FSH for 12 h. (Q) Quantitative analysis of Flag fluorescence intensity from (P). Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Knockdown, Expressing, Transfection, Over Expression, Construct, Plasmid Preparation, Fractionation, Western Blot, Marker, Immunofluorescence, Fluorescence

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: C646-mediated P300 inhibition inhibits mitochondrial biogenesis and follicular development in vivo. (A) Schematic diagram of the in vivo experimental procedure. Mice were randomly assigned to 5 groups: (1) control (DMSO/0.9% saline vehicle), (2) FSH alone, (3) FSH + C646 (15 mg/kg), (4) FSH + LMK-235 (15 mg/kg), and (5) FSH + SR-18292 (15 mg/kg). All intraperitoneal injections were administered at 12-h intervals. The FSH regimen followed a tapering protocol of 10 IU, 5 IU, and two 2-IU doses. The respective inhibitors were co-administered with each FSH injection. All drugs were dissolved in DMSO and diluted in 0.9% saline for administration. (B) Western blot analysis of Pan-Kla within histone regions and H4K5la levels following the indicated treatments in (A), with H4 used as a loading control for normalization. (C) Immunohistochemical detection of Pan-Kla expression following the indicated treatments in (A). Pan-Kla + normalized to total cell number. Scale bar, 200 μm. (D) qRT-PCR measurement of HDAC4 expression after specified treatments in (A). Tuba1a served as the loading control for data normalization. (E) Western blot assessment of HDAC4 expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (F) Co-IP assay assessing PGC-1α and pan-acetyl-lysine binding posttreatment in (A). For IP, PGC-1α acetylation was quantified as the ratio of acetylated to total PGC-1α. For Input, the levels of total acetylation and PGC-1α protein were normalized to TUBA1A. (G) Co-IP assay assessing PGC-1α and NRF1/2 binding posttreatment in (A). For IP, the binding of PGC-1α to NRF1/2 was measured by calculating the NRF1/2 to PGC-1α ratio. For Input, the levels of NRF1/2 and PGC-1α were normalized to TUBA1A. (H) qRT-PCR examination of Tfb1m , Tfb2m , and Tfam mRNA expression after the specified treatments in (A). Tuba1a served as the loading control for data normalization. (I) qRT-PCR was used to assess mitochondrial DNA copy number, specifically targeting the MT-CO2 and D-Loop regions, following the indicated treatments in (A). β-Actin served as the loading control for data normalization. (J) Western blot assessment of TOM20 expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (K) Using a radioimmunoassay (RIA), we quantified the serum estradiol (E2) concentrations across the treatment groups specified in (A). (L) Western blot assessment of CYP19A1 expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (M) Western blot assessment of proliferating cell nuclear antigen (PCNA) expression posttreatment in (A), with TUBA1A used as a loading control for normalization. (N) 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay detects the proliferation activity of mouse ovarian GCs following the indicated treatments in (A). EdU-positive cells normalized to total cell number. Scale bar, 100 μm. (O) Measurement of ovarian size following the indicated treatments in (A). (P) Measurement of ovarian weight following the indicated treatments in (A). The ovary weight was expressed relative to the body weight of the corresponding mouse. (Q) Measurement of follicle diameter following the indicated treatments in (A). (R) The counts of primary, secondary, and antral follicles were assessed via hematoxylin and eosin (H&E) staining as outlined in treatment (A). PF, primary follicle; SF, secondary follicle; AF, antral follicles. Scale bar, 500 μm. Data are presented as the mean ± SD from at least 3 independent experiments ( n ≥ 3). Statistical differences between groups were compared by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Inhibition, In Vivo, Control, Saline, Injection, Western Blot, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Binding Assay, RIA Assay, Activity Assay, Staining

Journal: Research

Article Title: Histone Lactylation Couples FSH-Driven Lactate Metabolism to Mitochondrial Biogenesis by Enhancing HDAC4-Mediated Deacetylation of PGC-1α in Granulosa Cells

doi: 10.34133/research.1045

Figure Lengend Snippet: Mechanistic model of H4K5la in FSH-driven mitochondrial biogenesis. FSH activates aerobic glycolysis in GCs, generating lactate that induces histone H4K5la via P300/CBP. This epigenetic modification promotes HDAC4 transcription, leading to HDAC4-mediated deacetylation of PGC-1α at K329/330. Deacetylated PGC-1α facilitates the recruitment of nuclear respiratory factors (NRF1 and NRF2) to promoter regions, initiating the expression of essential genes involved in mitochondrial biogenesis, such as TFAM , TFB1M , and TFB2M . This process ultimately leads to an expansion of the mitochondrial network. Through this lactate–H4K5la–HDAC4 axis, FSH synchronizes mitochondrial expansion with the bioenergetic demands of follicular development.

Article Snippet: Additional antibodies included P300 (86377S), CBP (7389S), PCNA (2586S), TOM20 (42406S), PGC-1α (2178S), and Flag (8146S) from Cell Signaling Technology; histone H4 (16047-1-AP), HDAC4 (66838-1-AP), NRF1 (66832-1-AP), GLUT1 (21829-1-AP), TUBA1A (11224-1-AP), ACAT1 (16215-1-AP), DLAT1 (13426-1-AP), and LDHA (21799-1-AP) from Proteintech; LDHB (PAB698Hu01) from Cloud-Clone Corp.; and NRF2 (PA5-27735) from Thermo Fisher Scientific.

Techniques: Modification, Expressing

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Representative HDAC6 PET tracers with IC 50 values for HDAC6.

Article Snippet: Recombinant human HDAC6, HDAC7, and HDAC1 were obtained from SignalChem Pharmaceutical Inc. (Richmond, BC, Canada), recombinant HDAC8 was obtained from Reaction Biology Corporation (Marvern, PA, USA), and recombinant human HDAC4 and mouse HDAC6 were prepared by Axcelead Drug Discovery Partners Inc. A test compound (1 μL) diluted with DMSO was added to 75 μL of assay buffer (24 mM Tris-HCl [pH 7.5], 135 mM NaCl, 0.35 mM KCl, 1 mM MgCl 2 , 0.01% Tween 20, 0.6 mM glutathione) in 384 well plates.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Affinity selection (AS)–MS binding study of HDAC6 with 16a and bavarostat. Data points are the mean ± standard error (SE) (four technical replicates). (A) Saturation binding curve. (B) Time course for the dissociation. (C) Binding parameters calculated from AS–MS data. Functional IC 50 values are shown for comparison.

Article Snippet: Recombinant human HDAC6, HDAC7, and HDAC1 were obtained from SignalChem Pharmaceutical Inc. (Richmond, BC, Canada), recombinant HDAC8 was obtained from Reaction Biology Corporation (Marvern, PA, USA), and recombinant human HDAC4 and mouse HDAC6 were prepared by Axcelead Drug Discovery Partners Inc. A test compound (1 μL) diluted with DMSO was added to 75 μL of assay buffer (24 mM Tris-HCl [pH 7.5], 135 mM NaCl, 0.35 mM KCl, 1 mM MgCl 2 , 0.01% Tween 20, 0.6 mM glutathione) in 384 well plates.

Techniques: Selection, Binding Assay, Functional Assay, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Brain and plasma concentration of 16a in wild-type (WT) and HDAC6 knockout (KO) mice. Compound 16a (1 mg/kg) was intravenously administered to WT and HDAC6 KO mice. Two hours after administration, the hippocampus and plasma were collected and the concentration of 16a in the brain (A) and plasma (B) was measured using liquid chromatography with tandem mass spectrometry (LC–MS/MS). The data are expressed in percentage of injected dose per gram of brain tissue (%ID/g) or per milliliter of plasma (%ID/mL), respectively, as mean ± standard error (SE), n = 4. ** P < 0.01, n.s.: not significant.

Article Snippet: Recombinant human HDAC6, HDAC7, and HDAC1 were obtained from SignalChem Pharmaceutical Inc. (Richmond, BC, Canada), recombinant HDAC8 was obtained from Reaction Biology Corporation (Marvern, PA, USA), and recombinant human HDAC4 and mouse HDAC6 were prepared by Axcelead Drug Discovery Partners Inc. A test compound (1 μL) diluted with DMSO was added to 75 μL of assay buffer (24 mM Tris-HCl [pH 7.5], 135 mM NaCl, 0.35 mM KCl, 1 mM MgCl 2 , 0.01% Tween 20, 0.6 mM glutathione) in 384 well plates.

Techniques: Clinical Proteomics, Concentration Assay, Knock-Out, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Injection

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Comparison of HDAC6 expression levels in a mouse and monkey using Western blotting. Hippocampus (Hip) was obtained from two mice, and HDAC6 expression was examined. Recombinant HDAC6 conjugated with GST (0.03, 0.1, 0.3, and 1 ng) was also used for the positive controls (A). Lysates of the hippocampus and corpus callosum (CC) obtained from a monkey were applied to Western blotting in duplicate. The same controls were used for comparison (B).

Article Snippet: Recombinant human HDAC6, HDAC7, and HDAC1 were obtained from SignalChem Pharmaceutical Inc. (Richmond, BC, Canada), recombinant HDAC8 was obtained from Reaction Biology Corporation (Marvern, PA, USA), and recombinant human HDAC4 and mouse HDAC6 were prepared by Axcelead Drug Discovery Partners Inc. A test compound (1 μL) diluted with DMSO was added to 75 μL of assay buffer (24 mM Tris-HCl [pH 7.5], 135 mM NaCl, 0.35 mM KCl, 1 mM MgCl 2 , 0.01% Tween 20, 0.6 mM glutathione) in 384 well plates.

Techniques: Comparison, Expressing, Western Blot, Recombinant

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Immunohistochemical (IHC) staining of HDAC6 protein in a monkey brain. Coronal brain sections, including the areas below were prepared from a monkey (a, putamen; b, caudate nucleus; c, corpus callosum; d, white matter; e, anterior cingulate cortex; f, thalamus; g, hippocampus; h, cerebellum; i, brain stem [pons]). They were immunostained with HDAC6 antibody.

Article Snippet: Recombinant human HDAC6, HDAC7, and HDAC1 were obtained from SignalChem Pharmaceutical Inc. (Richmond, BC, Canada), recombinant HDAC8 was obtained from Reaction Biology Corporation (Marvern, PA, USA), and recombinant human HDAC4 and mouse HDAC6 were prepared by Axcelead Drug Discovery Partners Inc. A test compound (1 μL) diluted with DMSO was added to 75 μL of assay buffer (24 mM Tris-HCl [pH 7.5], 135 mM NaCl, 0.35 mM KCl, 1 mM MgCl 2 , 0.01% Tween 20, 0.6 mM glutathione) in 384 well plates.

Techniques: Immunohistochemical staining, Immunohistochemistry