selective ezh2 methyltransferase inhibitor  (MedChemExpress)

Journal: Advanced Science

Article Title: Paternal Obesity‐Induced H3K27me3 Elevation Leads to MANF‐Mediated Transgenerational Metabolic Dysfunction in Female Offspring

doi: 10.1002/advs.202415956

Figure Lengend Snippet: Paternal obesity enhanced EZH2‐H3K27me3 modification, leading to transgenerational deregulation of the Manf gene in offspring. A) The protein levels of H3K9me3 and H3K27me3 in the liver of F0 male mice, as well as the protein levels of H3K27me3 in the liver of F1 and F2 female mice (n = 10 each group); B) ChIP‐RT‐qPCR detecting H3K27me3 binding to the promoter region of the Manf gene in liver tissue of F0 male mice and F1 female mice (n = 4 each group). Normalized to Input; C) The protein levels of H3K27me3 in the sperm of F0‐F2 male mice (n = 6 each group); D) The immunofluorescent staining of H3K27me3 (green) in 8‐cell embryo stage from F1‐F2 generation between CD and HFD groups (n = 8 each group). Nuclei are labeled by DAPI (blue). 200X. E) The mRNA levels of Ezh2 in the liver of F0 male mice and F1–F2 female mice (n = 10 each group); F) Protein levels of EZH2 in liver from F0 male mice and F1–F2 female mice between CD and HFD groups (n = 10 each group); (G) The protein levels of EZH2, H3K27me3, and MANF were measured in primary hepatocytes following treatment with a concentration gradient of DZNep (n = 3 each group); H) The protein levels of EZH2, H3K27me3, and MANF were measured in primary hepatocytes following treatment with a concentration gradient of GSK126 (n = 3 each group); I) The protein levels of EZH2, H3K27me3, and MANF in primary hepatocytes following treatment with or without PA, GSK126, or DZNep (n = 3 each group). Data are mean ± SEM. ns, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (two‐tailed t‐test or two‐way ANOVA).

Article Snippet: To simulate HFD stimulation in vitro and investigate the effect of palmitic acid (PA) on MANF expression, primary hepatocytes were seeded at 2–3 × 10 5 cells per well and treated with PA, which was dissolved in bovine serum albumin (BSA), and then diluted with complete medium to a final concentration of 0.1, 0.2, 0.3, and 0.4 mM for 48 h. To verify the time of PA‐induced changes in MANF, primary hepatocytes were treated with PA for 12, 24, and 48 h. To verify PA‐induced changes of ER stress and apoptosis, primary hepatocytes were treated with PA at 0.2 mM for 48 h. To simulate ER stress stimulation in vitro and gain more insight into the relationship of ER stress and the glucose metabolic dysfunction, primary hepatocytes were incubated with DMSO or Tg (10 −6 M), an ER stress inducer, for 48 h. To investigate the functions of EZH2, DNMT1, H3K27me3, and MANF, primary hepatocytes were treated with PA, GSK126 (S7061, Selleck, USA), a highly selective EZH2 methyltransferase inhibitor, 3‐deazaneplanocin A‐ (S7120, Selleck, USA), a competitive S‐adenosylhomocysteine hydroxylase (SAHH) inhibitor, which depletes EZH2 and the associated H3K27me3, or DC‐05, a DNMT1 inhibitor, for different concentrations at 48 h. [ ] DMSO was used as a negative control.

Techniques: Modification, Quantitative RT-PCR, Binding Assay, Staining, Labeling, Concentration Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Paternal Obesity‐Induced H3K27me3 Elevation Leads to MANF‐Mediated Transgenerational Metabolic Dysfunction in Female Offspring

doi: 10.1002/advs.202415956

Figure Lengend Snippet: Intraperitoneally injected DZNep in F0 male mice improves the liver histoarchitecture and reverses glucose metabolic dysfunction and ER stress, with altering H3K27me3 in the sperm and 8‐cell embryo stage. At the end of treatment with a DZNep with a dose of 1 mg kg −1 every 2 days for two weeks through intraperitoneal injection in F0 male mice. (A) IPGTT and AUC (n = 10 each group); B) IPITT and AUC (n = 10 each group); C) IPPTT (n = 10 each group); D) Fasting serum insulin levels (n = 10 each group); E) HOMA‐IR (n = 10 each group); F) The H&E, PAS, and Oil Red O staining (n = 3 each group). Fat vacuoles (blue arrows), infiltration of inflammatory cells (yellow arrows), and cell swelling (green arrows), glycogen accumulation (red arrows), triglyceride accumulation (purple arrows). NAS scores in different groups (n = 6 each group); G) The protein levels of H3K27me3 in the sperm of F0 male mice with or without treatment of DZNep (n = 6 each group); H) The immunofluorescent staining of H3K27me3 (green) in 8‐cell embryo stage of different groups (n = 8 each group). Nuclei are labeled by DAPI (blue). 200X; I,J) Protein levels of the EZH2‐H3K27me3‐MANF pathway, AKT, p‐AKT, p‐AKT/AKT, GSK3β, p‐GSK3β, p‐GSK3β/GSK3β, and the GRP78‐PERK‐EIF2α‐ATF4‐CHOP pathways in liver of different groups (n = 6 each group). Data are mean ± SEM. ns, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (one‐way or two‐way ANOVA).

Article Snippet: To simulate HFD stimulation in vitro and investigate the effect of palmitic acid (PA) on MANF expression, primary hepatocytes were seeded at 2–3 × 10 5 cells per well and treated with PA, which was dissolved in bovine serum albumin (BSA), and then diluted with complete medium to a final concentration of 0.1, 0.2, 0.3, and 0.4 mM for 48 h. To verify the time of PA‐induced changes in MANF, primary hepatocytes were treated with PA for 12, 24, and 48 h. To verify PA‐induced changes of ER stress and apoptosis, primary hepatocytes were treated with PA at 0.2 mM for 48 h. To simulate ER stress stimulation in vitro and gain more insight into the relationship of ER stress and the glucose metabolic dysfunction, primary hepatocytes were incubated with DMSO or Tg (10 −6 M), an ER stress inducer, for 48 h. To investigate the functions of EZH2, DNMT1, H3K27me3, and MANF, primary hepatocytes were treated with PA, GSK126 (S7061, Selleck, USA), a highly selective EZH2 methyltransferase inhibitor, 3‐deazaneplanocin A‐ (S7120, Selleck, USA), a competitive S‐adenosylhomocysteine hydroxylase (SAHH) inhibitor, which depletes EZH2 and the associated H3K27me3, or DC‐05, a DNMT1 inhibitor, for different concentrations at 48 h. [ ] DMSO was used as a negative control.

Techniques: Injection, Staining, Labeling

Journal: Advanced Science

Article Title: Paternal Obesity‐Induced H3K27me3 Elevation Leads to MANF‐Mediated Transgenerational Metabolic Dysfunction in Female Offspring

doi: 10.1002/advs.202415956

Figure Lengend Snippet: PA enhanced the activation of the EZH2‐H3K27me3‐MANF pathway, leading to ER stress, glucose homeostasis dysfunction and apoptosis in vitro. A) PAS staining of primary hepatocytes in different groups (n = 3 each group); B) Tg‐mediated calcium content of primary hepatocytes in different groups (cell number = 15 per group); C) Representative immunofluorescent staining of EZH2 (green) and DNMT1 (red) in primary hepatocytes of different groups (n = 3 each group). Nuclei are labeled by DAPI (blue). Scale bar: 50 µm; D) Apoptotic cells in primary hepatocytes of different groups were identified by using CytoFLEX (n = 6 each group); E) Representative WB of the EZH2‐H3K27me3‐MANF pathway, AKT, p‐AKT, p‐AKT/AKT, GSK3β, p‐GSK3β, p‐GSK3β/GSK3β, and the GRP78‐PERK‐EIF2α‐ATF4‐CHOP pathways in primary hepatocytes of different groups; F) Protein levels of the EZH2‐H3K27me3‐MANF pathway in primary hepatocytes of different groups (n = 6 each group); G) Protein levels of the GRP78‐PERK‐EIF2α‐ATF4‐CHOP pathways in primary hepatocytes of different groups (n = 6 each group); H) Protein levels of AKT, p‐AKT, p‐AKT/AKT, GSK3β, p‐GSK3β, p‐GSK3β/GSK3β in primary hepatocytes of different groups (n = 6 each group); Data are mean ± SEM. ns, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (two‐tailed t‐test or two‐way ANOVA).

Article Snippet: To simulate HFD stimulation in vitro and investigate the effect of palmitic acid (PA) on MANF expression, primary hepatocytes were seeded at 2–3 × 10 5 cells per well and treated with PA, which was dissolved in bovine serum albumin (BSA), and then diluted with complete medium to a final concentration of 0.1, 0.2, 0.3, and 0.4 mM for 48 h. To verify the time of PA‐induced changes in MANF, primary hepatocytes were treated with PA for 12, 24, and 48 h. To verify PA‐induced changes of ER stress and apoptosis, primary hepatocytes were treated with PA at 0.2 mM for 48 h. To simulate ER stress stimulation in vitro and gain more insight into the relationship of ER stress and the glucose metabolic dysfunction, primary hepatocytes were incubated with DMSO or Tg (10 −6 M), an ER stress inducer, for 48 h. To investigate the functions of EZH2, DNMT1, H3K27me3, and MANF, primary hepatocytes were treated with PA, GSK126 (S7061, Selleck, USA), a highly selective EZH2 methyltransferase inhibitor, 3‐deazaneplanocin A‐ (S7120, Selleck, USA), a competitive S‐adenosylhomocysteine hydroxylase (SAHH) inhibitor, which depletes EZH2 and the associated H3K27me3, or DC‐05, a DNMT1 inhibitor, for different concentrations at 48 h. [ ] DMSO was used as a negative control.

Techniques: Activation Assay, In Vitro, Staining, Labeling, Two Tailed Test