anti dnmt1  (Novus Biologicals)

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A) When the concentration of 5-aza-dC reached 15 μM, cellular viability of hBMSCs from the ONFH group reached its peak at 24, 48, and 72 h (n = 6). (B) The cellular proliferative capacity was evaluated in hBMSCs of the control group, ONFH group, and 5-aza-dC treated group at 24, 48, and 72 h (n = 6). (C) qRT-PCR analysis revealed the relative expressions of H19 and DNMTs in undifferentiated hBMSCs of the control group, ONFH group, and 5-aza-dC treated group (n = 10). (D) Schematic diagram showed the location of 12 CpG sites within the analyzed region of the H19 promoter CpG island. (E) The BSP assay was used to analyze the methylation status of these CpG sites in the H19 promoter in hBMSCs of the three group.In the resulting methylation graph, each column represents a single individual subject sample, and each row corresponds to one of the 12 specific CpG sites analyzed, ordered by their genomic location. Empty dots: unmethylated CpGs; black dots: methylated CpGs. (F–I) qRT-PCR analysis showed the relative expressions of H19 (F) , Dnmt1 (G) , Dnmt3a (H) , and Dnmt3b (I) at days 3, 7, and 14 during osteogenic differentiation (n = 10). (J–M) qRT-PCR analysis showed the relative expressions of H19 (J) , Dnmt1 (K) , Dnmt3a (L) , and Dnmt3b (M) at days 3, 7, and 14 during adipogenic differentiation (n = 10).Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Concentration Assay, Control, Quantitative RT-PCR, PCR-BSP Assay, Methylation

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A–F) Representative western blot bands and relative quantification of proteins indicated that Dnmt1 siRNA (A–B) , Dnmt3a siRNA (C–D) , and Dnmt3b siRNA (E–F) efficiently decreased the protein levels of Dnmt1 , Dnmt3a and Dnmt3b in hBMSCs of the ONFH group. β-actin was used as a loading control. (G) qRT-PCR analysis showed the relative expression of H19 in hBMSCs after DNMT knockdown. (H) BSP analysis was used to detect the methylation status of the H19 promoter. (I–J) Representative western blot images and quantification of relative protein expression indicated that overexpression of Dnmt1 by transfection efficiently increased Dnmt1 expression in hBMSCs. β-actin was used as a loading control. (K) qRT-PCR analysis showed the expression differences of H19 in hBMSCs from three different treatment groups. (L) BSP analysis was used to detect the methylation status of the H19 promoter in hBMSCs of the three groups. Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, n = 3 per group, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control; si/siRNA: small interfering RNA; oe: overexpression.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Western Blot, Quantitative Proteomics, Control, Quantitative RT-PCR, Expressing, Knockdown, Methylation, Over Expression, Transfection, Negative Control, Small Interfering RNA

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A–B) ARS staining (A) and quantification (B) were performed to measure the calcium deposits in hBMSCs after 14 days of induction of osteogenic differentiation. Scale bars: 100 μm. (C–D) ORO staining (C) and quantification (D) were used to evaluate the intracellular lipid accumulation in hBMSCs after 21 days of induction of adipogenic differentiation. Scale bars: 50 μm. (E–G) After DNMT knockdown, the representative western blot band (E) and quantitative analysis showed the relative expressions of RUNX2 and COL1A1 (F) , as well as FABP4 and PPARγ (G) . β-actin was used as the loading control. (H–I) ARS staining (H) and quantification (I) were performed to measure the calcium deposits in hBMSCs. Scale bars: 100 μm. (J-K) ORO staining (J) and quantification (K) were used to evaluate the intracellular lipid accumulation in hBMSCs. Scale bars: 50 μm. (L–N) After overexpression of Dnmt1 , the representative western blot band (L) and quantitative analysis showed the relative expressions of RUNX2 and COL1A1 (M) as well as FABP4 and PPARγ (N) . β-actin was used as the loading control. Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, n = 3 per group, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. NC, negative control; si/siRNA: small interfering RNA; oe: overexpression.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Staining, Knockdown, Western Blot, Control, Over Expression, Negative Control, Small Interfering RNA

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: (A) Schematic diagram of the experimental design for investigating the effects of implantation with Dnmt1 -knockdown or H19 -overexpression rBMSCs on the femoral heads of MPS-treated rats. (B) Fluorescence microscopy was used to observe infection efficiency in primary rBMSCs. Scale bars: 100 μm. (C) Western blot analysis showed that Dnmt1 shRNA efficiently decreased the protein expression levels of Dnmt1 in rBMSCs of MPS-treated rats. β-actin was used as a loading control. (D) Representative micro-CT images of the femoral head of each treatment group at week 6 after rBMSC transplantation are shown. (E–H) Micro-CT quantitative results are expressed as BV/TV (E) , Tb.Th (F) , Tb.N (G) , and Tb.Sp (H) (n = 6). (I) Representative images of H&E staining for each treatment group at week 6 after rBMSC implantation were shown and osteonecrosis was characterized by the empty lacunae (black arrow) or pyknotic nucleus of osteocytes (blue arrow) in trabecular bone. Scale bars: 25 μm. (J–K) Representative IHC images and quantitative analysis of positive stain of COL1A1 in the femoral head of each treatment group were shown (n = 6). Scale bars: 25 μm. (L–M) Representative IHC images and quantitative analysis of positive stain of FABP4 in the femoral head of each treatment group are shown (n = 6). Scale bars: 25 μm. Statistical analysis was performed using one/two-way ANOVA with Bonferroni’s post-hoc test. Data are presented as mean ± SD, #P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC: normal control, sh/shRNA: short hairpin RNA, sh-Ctrl: control shRNA.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: Knockdown, Over Expression, Fluorescence, Microscopy, Infection, Western Blot, shRNA, Expressing, Control, Micro-CT, Transplantation Assay, Staining

Journal: PLOS One

Article Title: Glucocorticoid-Induced alterations in DNA methylation in the H19 promoter of Bone Marrow-Derived Mesenchymal Stem Cells are associated with the pathogenesis of osteonecrosis

doi: 10.1371/journal.pone.0345372

Figure Lengend Snippet: A schematic diagram summarizing the proposed role of the Dnmt1/H19/GSK-3β axis in the pathogenesis of GC-induced ONFH.

Article Snippet: A short hairpin RNA (shRNA) targeting Dnmt1 was synthesized and cloned into a pLVX vector (Cyagen, Guangzhou, China) for the knockdown of Dnmt1 in rat BMSCs (rBMSCs).

Techniques: