dnmt3a rabbit polyclonal antibody  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells

doi: 10.1016/j.redox.2020.101716

Figure Lengend Snippet: Hypermethylation of the SOD2 upstream regulatory region is dependent on DNMT3A. (A) Location and methylation status of six CpG sites in the SOD2 upstream regulatory region analysed by bisulphite sequencing (BSP) in control and DGCR8 knockdown hMSCs (n = 12 individual clones). Each circle represents a single sequencing reaction of each CpG site (white circles, unmethylated CpG sites; black circles, methylated CpGs). (B) Total ROS levels are measured by H 2 DCF-DA after 5-aza-dC treatment (2 μM) in DGCR8 knockdown hMSCs. Error bars denote the standard error of the mean of three independent experiments (** P < 0.01). (C) Immunoblot showing the levels of SOD2 and Ras signalling components after 5-aza-dC treatment (2 μM) in DGCR8 knockdown hMSCs. (D) Mitochondrial membrane potential (MMP) was assessed by JC-1 staining in DGCR8 knockdown hMSCs. Representative fluorescence microscopy images of siGFP- or siDGCR8-transfected cells, as well as 5-aza-dC-treated DGCR8 knockdown cells. Scale bars, 200 μm. (E) The mRNA levels of DNMTs were measured by quantitative real-time PCR. Data were normalised to GAPDH mRNA levels. Error bars indicate the standard error of the mean of three independent experiments (* P < 0.05). (F) Western blot analysis of DNMT3A protein levels in DGCR8 knockdown hMSCs.

Article Snippet: After electrophoresis, proteins were transferred onto nitrocellulose membranes (GE Healthcare) and incubated overnight at 4 °C with appropriate primary antibodies: H-Ras rabbit polyclonal antibody (sc-520, Santa Cruz; 1:200), MEK1/2 (D1A5) rabbit monoclonal antibody (8727; Cell Signalling; 1:1000), phospho-Erk1/2 mouse monoclonal antibody (9106, Cell Signalling; 1:1000), Erk1/2 rabbit polyclonal antibody (9102, Cell Signalling; 1:1000), phospho-p53 (Ser15) rabbit antibody (9284, Cell Signalling; 1:500), p53 rabbit polyclonal antibody (sc-6243, Santa Cruz; 1:1000), p21 rabbit monoclonal antibody (2947, Cell Signalling; 1:1000), SOD1 rabbit polyclonal antibody (sc-11407, Santa Cruz; 1:1000), SOD2 mouse monoclonal antibody (611,580, BD Biosciences; 1:1000), SOD3 mouse monoclonal antibody (ab28442, Abcam; 1:2000), DNMT3A rabbit polyclonal antibody (2160, Cell Signalling; 1:1000), alpha-tubulin rabbit antibody (SAB3501071, Sigma Aldrich; 1:5000), and alpha-tubulin mouse antibody (T9026, Sigma Aldrich; 1:5000).

Techniques: Methylation, Bisulfite Sequencing, Control, Knockdown, Clone Assay, Sequencing, Western Blot, Membrane, Staining, Fluorescence, Microscopy, Transfection, Real-time Polymerase Chain Reaction

Journal: Redox Biology

Article Title: Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells

doi: 10.1016/j.redox.2020.101716

Figure Lengend Snippet: miR-29a-3p and miR-30c-5p directly repress DNMT3A and rescue oxidative stress and premature senescence induced by DGCR8 knockdown. (A) Schematic illustration of miR-30c-5p (site 1) and miR-29a-3p (site 2) binding sites within the human DNMT3A 3′ UTR (662–868) region, which was cloned downstream of the Renilla luciferase reporter gene in the psiCHECK-2 vector. Two predicted targeting sites and the corresponding mutations are listed. The mutagenised nucleotides are underlined and highlighted in red. The construct for the mutant 3′ UTR contained both mutagenised sites (M1+M2). Data were normalised to firefly luciferase. (B) Luciferase reporter assay. 293T cells were co-transfected with luciferase reporters carrying either the wild-type DNMT3A 3′ UTR (Dnmt3a) or the mutagenised DNMT3A 3′ UTR (mDnmt3a), as well as 50 nM negative control mimic (miR-Control) or miR-29a-3p or/and miR-30c-5p mimics. Error bars indicate the standard error of the mean of three experiments. (** P < 0.01 and *** P < 0.001 compared to the miR-control). (C) The mRNA and protein expression levels of DNMT3A in DGCR8 knockdown hMSCs with or without miR-29a-3p or/and miR-30c-5p overexpression. The mRNA levels of DNMT3A were normalised to GAPDH . Error bars denote the standard error of the mean of three independent experiments (top). Immunoblot showing DNMT3A protein levels in DGCR8 knockdown cells with or without miR-29a-3p or/and miR-30c-5p forced expression (bottom). (D) Total ROS levels in DGCR8 knockdown hMSCs after miR-29a-3p or/and miR-30c-5p overexpression were measured using H 2 DCF-DA. Error bars denote the standard error of the mean of three independent experiments (** P < 0.01). Immunoblot showing the protein levels of SOD2 and Ras signalling components (bottom). (E) Mitochondrial membrane potential (MMP) in DGCR8 knockdown hMSCs after miR-29a-3p or/and miR-30c-5p overexpression was determined using JC-1. Representative fluorescence microscopy images in siGFP, siDGCR8, and mimic-transfected siDGCR8 hMSCs. Scale bar, 100 μm. (F) Western blot showing the protein levels of tumour suppressors and senescence markers, including p21, p53, and p-p53 (ser15), after miR-29a-3p or/and miR-30c-5p overexpression in DGCR8 knockdown hMSCs. (G) Mean growth rate of siGFP and siDGCR8 hMSCs co-transfected with miR-29a-3p or/and miR-30c-5p mimics, as determined by MTS. Error bars denote the standard error of the mean of three independent experiments (** P < 0.01).

Article Snippet: After electrophoresis, proteins were transferred onto nitrocellulose membranes (GE Healthcare) and incubated overnight at 4 °C with appropriate primary antibodies: H-Ras rabbit polyclonal antibody (sc-520, Santa Cruz; 1:200), MEK1/2 (D1A5) rabbit monoclonal antibody (8727; Cell Signalling; 1:1000), phospho-Erk1/2 mouse monoclonal antibody (9106, Cell Signalling; 1:1000), Erk1/2 rabbit polyclonal antibody (9102, Cell Signalling; 1:1000), phospho-p53 (Ser15) rabbit antibody (9284, Cell Signalling; 1:500), p53 rabbit polyclonal antibody (sc-6243, Santa Cruz; 1:1000), p21 rabbit monoclonal antibody (2947, Cell Signalling; 1:1000), SOD1 rabbit polyclonal antibody (sc-11407, Santa Cruz; 1:1000), SOD2 mouse monoclonal antibody (611,580, BD Biosciences; 1:1000), SOD3 mouse monoclonal antibody (ab28442, Abcam; 1:2000), DNMT3A rabbit polyclonal antibody (2160, Cell Signalling; 1:1000), alpha-tubulin rabbit antibody (SAB3501071, Sigma Aldrich; 1:5000), and alpha-tubulin mouse antibody (T9026, Sigma Aldrich; 1:5000).

Techniques: Knockdown, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, Mutagenesis, Reporter Assay, Transfection, Negative Control, Control, Expressing, Over Expression, Western Blot, Membrane, Fluorescence, Microscopy