Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 5. 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).

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 × 105 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.[39a] DMSO was used as a negative control.

Techniques: Quantitative RT-PCR, Binding Assay, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 7. 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 × 105 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.[39a] DMSO was used as a negative control.

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