Journal: BMC Molecular Biology

Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

doi: 10.1186/1471-2199-15-24

Figure Lengend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

Article Snippet: Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

Techniques: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Western Blot, Autoradiography

Journal: Frontiers in Pharmacology

Article Title: Attenuation of High Glucose-Induced Damage in RPE Cells through p38 MAPK Signaling Pathway Inhibition

doi: 10.3389/fphar.2021.684680

Figure Lengend Snippet: Effects of DMF on p38 MAPK pathway. ARPE-19 cells exposed to normal glucose (NG) or high glucose (HG) w/or w/o DMF (10 μM) for 24 h (A) p38 MAPK was measured by western blot analysis; band densities were quantified by ImageQuantTL software and normalized values were plotted in the histogram shown on the right (*** p < 0.001 vs. NG; † p < 0.05 vs. untreated/HG; n = 6). (B) ARPE-19 cells exposed to normal glucose (NG) or high glucose (HG) were pretreated with SB202190 (10 µM), a p38 MAPK inhibitor for 30 min and then treated with DMF (10 μM) for 24 h. VEGF levels were measured through ELISA. * p < 0.05 vs. NG untreated cells; n = 6. ( C ) MTT analysis: values are expressed as percentage of cell viability in NG untreated cells. * p < 0.05 or ** p < 0.01 vs. NG untreated cells; n = 6.

Article Snippet: Primary antibodies: BAX (sc-493, Santa Cruz Biotechnology, Inc, CA, United States), Bcl-2 (sc-509, Santa Cruz Biotechnology, Inc, CA, United States), COX-2 (sc-19999, Santa Cruz Biotechnology, Inc, CA, United States), iNOS (sc-651, Santa Cruz Biotechnology, Inc, CA, United States), p38 MAPK (#9212, Cell Signaling Technology, United States), p-p38 MAPK (Thr180/Tyr182) (#9211, Cell Signaling Technology, United States) and β -tubulin (sc-9104, Santa Cruz Biotechnology, Inc, CA, United States).

Techniques: Western Blot, Software, Enzyme-linked Immunosorbent Assay