Journal: International Journal of Molecular Sciences

Article Title: Targeted DNA Methylation Using Modified DNA Probes: A Potential Therapeutic Tool for Depression and Stress-Related Disorders

doi: 10.3390/ijms26125643

Figure Lengend Snippet: Dose-dependent DNAm and gene expression changes following transfection of methylated DNA probes against FKBP5 . ( A ) 293HEK cells were transfected with a single-stranded unmethylated probe (500 ng − SssI), a single-stranded methylated probe at two different concentrations (250 ng and 500 ng + SssI), or a double-stranded methylated probe (500 ng + SssI +DS). Untransfected cells served as controls (Control). DNAm levels of five CpG sites at the conserved glucocorticoid response element (GRE) of human FKBP5 intron 5 were analyzed by bisulfite pyrosequencing. Data for the first two CpGs are shown. ( B ) Typical pyrograms obtained from bisulfite pyrosequencing are shown for each group at CpG-1. The percent DNAm determination occurs when R (or A/G) is dispensed, and it corresponds to the reverse complement of T/C (T for unmethylated and C for methylation CpG, respectively). Each pyrogram represents % methylation from one sample. ( C ) FKBP5 expression was measured by qRT-PCR in the same groups of 293HEK cells as in ( A ) treated with 1 μM dexamethasone (DEX) for four hours prior to collection. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t -tests were performed with n = 4 per group. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: Human embryonic kidney 293 (293HEK) and mouse pituitary AtT-20 cells were purchased from Atcc.org and were treated with 1 μM dexamethasone (DEX) for 5 days and cultured for an additional 5 days without any DEX.

Techniques: Gene Expression, Transfection, Methylation, Control, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Targeted DNA Methylation Using Modified DNA Probes: A Potential Therapeutic Tool for Depression and Stress-Related Disorders

doi: 10.3390/ijms26125643

Figure Lengend Snippet: Persistence and accumulation of DNA methylation. ( A ) 293HEK cells were transfected with the unmethylated (−SssI) and methylated (+SssI) FKBP5 DNA probe at Week 1 (W1), after which the cells were cultured for an additional four weeks (W4) before analysis by bisulfite pyrosequencing. ( B ) 293HEK cells were transfected with unmethylated (−SssI) and methylated (+SssI) FKBP5 DNA probes consecutively every three days and expanded, while 50% of the cells were collected for analysis. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t -tests were performed with n = 4 per group. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: Human embryonic kidney 293 (293HEK) and mouse pituitary AtT-20 cells were purchased from Atcc.org and were treated with 1 μM dexamethasone (DEX) for 5 days and cultured for an additional 5 days without any DEX.

Techniques: DNA Methylation Assay, Transfection, Methylation, Cell Culture, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Targeted DNA Methylation Using Modified DNA Probes: A Potential Therapeutic Tool for Depression and Stress-Related Disorders

doi: 10.3390/ijms26125643

Figure Lengend Snippet: DNAm and gene expression analysis following transfection of methylated DNA probes against MAOA . ( A ) 293HEK cells were transfected with a single-stranded unmethylated probe (-SssI) or a single-stranded methylated probe (+SssI). DNAm levels of 14 CpG sites at a regulatory region of human MAOA were analyzed by bisulfite pyrosequencing. ( B ) MAOA expression was measured by qRT-PCR in the same groups of 293HEK cells. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t -tests were performed with n = 4 per group. * p < 0.05 and ** p < 0.01. A one-way ANOVA was used to test for overall differences across all 14 CpGs, followed by Tukey’s post hoc test for pairwise comparisons.

Article Snippet: Human embryonic kidney 293 (293HEK) and mouse pituitary AtT-20 cells were purchased from Atcc.org and were treated with 1 μM dexamethasone (DEX) for 5 days and cultured for an additional 5 days without any DEX.

Techniques: Gene Expression, Transfection, Methylation, Expressing, Quantitative RT-PCR, Two Tailed Test