Journal: Cells

Article Title: D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation

doi: 10.3390/cells11071230

Figure Lengend Snippet: Effect of Na 2 S on the amounts of Nrf2 and CMA-related proteins in AD293 cells. ( A ) Immunoblot analyses of Nrf2 and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 4 and 24 h. ( B ) Quantitative analyses of Nrf2 amounts from the immunoblot results shown in A. ( C ) Immunoblot analyses of CMA-related proteins (LAMP2A, Hsc70) and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 24 h. ( D ) Quantitative analyses of LAMP2A and Hsc70 amounts from the immunoblot results shown in C. ( E ) Immunoblot analyses of a CMA/mA substrate (MEF2D) and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 24 h. ( F ) Quantitative analysis of MEF2D amount from the immunoblot results shown in F. Whole blot images are presented in . Amounts of β-actin were used as internal controls for quantification. Numbers in the columns represent the number of samples. * p < 0.05, *** p < 0.001 (unpaired t -test).

Article Snippet: Anti-myocyte enhancer factor 2D (MEF2D) mouse monoclonal antibody (#610774) was obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Western Blot

Journal: Cells

Article Title: D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation

doi: 10.3390/cells11071230

Figure Lengend Snippet: Effect of long-term treatment with D-cysteine on the amounts of Nrf2- and CMA-related proteins in cerebellar lysates from ICR mice. ( A ) Immunoblot analyses of Nrf2, NQO1, LAMP2A, Hsc70, MEF2D, and β-actin in cerebellar lysates from ICR mice daily treated with saline (Sal) and D-cysteine (100 mg/kg/day) for 10 weeks. Whole blot images are presented in . ( B ) Quantitative analyses of the amounts of Nrf2, NQO1, LAMP2A, Hsc70, and MEF2D shown in A. Amounts of β-actin were used as internal controls for the quantification. * p < 0.05, ** p < 0.01 vs. saline-treated mice (unpaired t -test, n = 5 in both saline- and D-cysteine-treated mice).

Article Snippet: Anti-myocyte enhancer factor 2D (MEF2D) mouse monoclonal antibody (#610774) was obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Western Blot

Journal: Cellular & molecular biology letters

Article Title: Maternal high-fat diet orchestrates offspring hepatic cholesterol metabolism via MEF2A hypermethylation-mediated CYP7A1 suppression.

doi: 10.1186/s11658-024-00673-8

Figure Lengend Snippet: Fig. 4 Maternal high-fat feeding upregulates DNMTs expression and downregulates MEF2A transcription factor in offspring. A mRNA expression of Dnmts and Tets. B Representative immunoblot images for DNMTs. C Immunoblot analysis of DNMT1, DNMT3A, and DNMT3B. D Venn diagram of predicted transcription factors and DEGs. E mRNA expression of candidate transcription factors. F Representative immunoblot images for MEF2A. G Immunoblot analysis of MEF2A. H Representative immunohistochemistry images for MEF2A. I Immunohistochemistry analysis of MEF2A. The scale bar indicates 50 μm. F1, the second generation (offspring); Ctrl, standard diet; HFD, high-fat diet; Dnmts, DNA methyltransferases; Tets, ten-eleven translocation dioxygenases; MEF2A, myocyte enhancer factor 2A; DEGs, differentially expressed genes. Data presented as the mean ± SEM. *P < 0.05, ***P < 0.001 versus F1-Ctrl group. n = 5–8 litters for F1-Ctrl group, n = 5–8 litters for F1-HFD group, one male offspring per litter

Article Snippet: For immunohistochemistry, liver sections were pretreated in citrate buffer (PH 6.0, 98 °C) for 20 min, followed by quenching of endogenous peroxidase activity with 0.3% H2O2 for 15 min. After blocking in goat serum (C0265, Beyotime, Beijing, China) for 20 min, sections were incubated with primary antibodies against myocyte enhancer factor 2A (MEF2A) (1:1000, ab264329, Abcam, Cambridge, UK) and CYP7A1 (1:1000, 18054– 1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunohistochemistry, Translocation Assay

Journal: Cellular & molecular biology letters

Article Title: Maternal high-fat diet orchestrates offspring hepatic cholesterol metabolism via MEF2A hypermethylation-mediated CYP7A1 suppression.

doi: 10.1186/s11658-024-00673-8

Figure Lengend Snippet: Fig. 5 MEF2A regulates cholesterol metabolism via targeting CYP7A1. A mRNA expression of Mef2a. B Representative immunoblot images for MEF2A and CYP7A1. C mRNA expression of Cyp7a1. D Immunoblot analysis of CYP7A1. E Representative images of Oil Red O staining of HepG2 cells. F mRNA expression of Mef2a. G; Representative immunoblot images for MEF2A and CYP7A1. H mRNA expression of Cyp7a1. I Immunoblot analysis of CYP7A1. J Representative images of Oil Red O staining of HepG2 cells. K Predicted MEF2A transcription factor binding sites in Cyp7a1 promoter. L Relative luciferase activity of dual luciferase assay. The scale bar indicates 50 μm. Ctrl, control; siMEF2A, down-regulation of MEF2A; oeMEF2A, over-expression of MEF2A; MEF2A, myocyte enhancer factor 2A; CYP7A1, cholesterol 7α-hydroxylase. Data presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl n = 3–5

Article Snippet: For immunohistochemistry, liver sections were pretreated in citrate buffer (PH 6.0, 98 °C) for 20 min, followed by quenching of endogenous peroxidase activity with 0.3% H2O2 for 15 min. After blocking in goat serum (C0265, Beyotime, Beijing, China) for 20 min, sections were incubated with primary antibodies against myocyte enhancer factor 2A (MEF2A) (1:1000, ab264329, Abcam, Cambridge, UK) and CYP7A1 (1:1000, 18054– 1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Western Blot, Staining, Binding Assay, Luciferase, Activity Assay, Control, Over Expression

Journal: Cellular & molecular biology letters

Article Title: Maternal high-fat diet orchestrates offspring hepatic cholesterol metabolism via MEF2A hypermethylation-mediated CYP7A1 suppression.

doi: 10.1186/s11658-024-00673-8

Figure Lengend Snippet: Fig. 6 Maternal high-fat diet feeding induces hepatic mef2a hypermethylation in offspring. A CpG sites measured in Mef2a promoter. B DNA methylation levels of different CpG sites in Mef2a promoter. C Average DNA methylation level in Mef2a promoter. D Correlation between Mef2a methylation level and Mef2a mRNA expression. E Correlation between Mef2a methylation level and MEF2A protein expression. F mRNA expression of Mef2a in 5-AZA-treated HepG2 cells. G Representative immunoblot images for MEF2A in 5-AZA-treated HepG2 cells. H Immunoblot analysis of MEF2A in 5-AZA-treated HepG2cells. I Relative luciferase activity of methylated or unmethylated plasmids in H9C2 cells. F1, the second generation (offspring); Ctrl, standard diet; HFD, high-fat diet; MEF2A, myocyte enhancer factor 2A; 5-AZA, 5-azacitidine. Data presented as the mean ± SEM. *P < 0.05, ***P < 0.001 versus F1-Ctrl group. n = 7–8 litters/group, one male offspring per litter. *P < 0.05, ***P < 0.001 versus Ctrl. n = 6–7 litters for F1-Ctrl group, n = 6–7 litters for F1-HFD group, one male offspring per litter

Article Snippet: For immunohistochemistry, liver sections were pretreated in citrate buffer (PH 6.0, 98 °C) for 20 min, followed by quenching of endogenous peroxidase activity with 0.3% H2O2 for 15 min. After blocking in goat serum (C0265, Beyotime, Beijing, China) for 20 min, sections were incubated with primary antibodies against myocyte enhancer factor 2A (MEF2A) (1:1000, ab264329, Abcam, Cambridge, UK) and CYP7A1 (1:1000, 18054– 1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: DNA Methylation Assay, Methylation, Expressing, Western Blot, Luciferase, Activity Assay

Journal: Cellular & molecular biology letters

Article Title: Maternal high-fat diet orchestrates offspring hepatic cholesterol metabolism via MEF2A hypermethylation-mediated CYP7A1 suppression.

doi: 10.1186/s11658-024-00673-8

Figure Lengend Snippet: Fig. 7 Maternal high-fat diet feeding contributes to hepatic Mef2a hypermethylation in fetus. A mRNA expression of Mef2a in fetal liver. B Representative immunoblot images for MEF2A in fetal liver. C Immunoblot analysis of MEF2A in fetal liver. D,E Average DNA methylation level in Mef2a promoter. F–H CYP7A1 mRNA and protein expression in the fetal liver. F1, the second generation (offspring); Ctrl, standard diet; HFD, high-fat diet; E18.5, embryonic day 18.5; MEF2A, myocyte enhancer factor 2A; CYP7A1, cholesterol 7α-hydroxylase. Data presented as the mean ± SEM. *P < 0.05 versus F1-Ctrl. n = 5–6 litters for F1-Ctrl group, n = 5–7 litters for F1-HFD group, one male offspring per litter

Article Snippet: For immunohistochemistry, liver sections were pretreated in citrate buffer (PH 6.0, 98 °C) for 20 min, followed by quenching of endogenous peroxidase activity with 0.3% H2O2 for 15 min. After blocking in goat serum (C0265, Beyotime, Beijing, China) for 20 min, sections were incubated with primary antibodies against myocyte enhancer factor 2A (MEF2A) (1:1000, ab264329, Abcam, Cambridge, UK) and CYP7A1 (1:1000, 18054– 1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Western Blot, DNA Methylation Assay

Journal: Cellular & molecular biology letters

Article Title: Maternal high-fat diet orchestrates offspring hepatic cholesterol metabolism via MEF2A hypermethylation-mediated CYP7A1 suppression.

doi: 10.1186/s11658-024-00673-8

Figure Lengend Snippet: Fig. 8 Summary figure. Schematic diagram illustrating the mechanisms of maternal high-fat diet feeding in hepatic lipid accumulation in offspring mice in the study. Maternal high-fat diet can mediate DNA methylation changes in Mef2a, leading to suppression of hepatic CYP7A1 expression and subsequent hepatic cholesterol accumulation in offspring at weaning age. MEF2A, myocyte enhancer factor 2A; CYP7A1, cholesterol 7α-hydroxylase

Article Snippet: For immunohistochemistry, liver sections were pretreated in citrate buffer (PH 6.0, 98 °C) for 20 min, followed by quenching of endogenous peroxidase activity with 0.3% H2O2 for 15 min. After blocking in goat serum (C0265, Beyotime, Beijing, China) for 20 min, sections were incubated with primary antibodies against myocyte enhancer factor 2A (MEF2A) (1:1000, ab264329, Abcam, Cambridge, UK) and CYP7A1 (1:1000, 18054– 1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: DNA Methylation Assay, Expressing