Journal: Scientific Reports

Article Title: Loss of Dnmt3a induces CLL and PTCL with distinct methylomes and transcriptomes in mice

doi: 10.1038/srep34222

Figure Lengend Snippet: (a) Percentage of Dnmt3a Δ/Δ mice diagnosed with CLL (red), PTCL (blue), or mixed CLL/PTCL (green) at time of harvest, as determined FACS. N = 28. (b) Immunoblot analysis of Dnmt3b and Dnmt1 proteins in Dnmt3a +/+ normal thymus (Th) and spleen (SP), Dnmt3a Δ/Δ PTCL, and Dnmt3a Δ/Δ CLL samples. Dnmt3b −/− (3b KO) cells were used as a negative control. HDAC1 is shown as a loading control. (c) Representative FACS diagram showing CD19 and CD5 expression in EGFP- (black) and EGFP+ (green) cellular populations isolated from the spleen of a lethally irradiated FVB recipient mouse injected with bone marrow Dnmt3a Δ/Δ bone marrow. The mouse was harvested 8 weeks post injection. Percentage of cells in each quadrant are shown in the top right in red. (d) Kaplan-Meier survival curves for FVB mice lethally irradiated and injected with Dnmt3a +/+ or Dnmt3a Δ/Δ (N = 8) bone marrow cells. (e) Representative FACS diagram showing CD19 and CD5 expression in EGFP- (black) and EGFP+ (green) cell isolated from the spleen, blood and bone marrow of a lethally irradiated FVB recipient mice injected with Dnmt3a Δ/Δ bone marrow. The mouse was harvested 9 months post injection when terminally ill. Percentage of cells staining positive in each quadrant are shown in the top right in red. (f) Percentage of lethally irradiated wild-type mice injected with Dnmt3a Δ/Δ bone marrow cell that were diagnosed with CLL (red) or MBL (blue) at time of harvest, as determined FACS. N = 8. (g) Percentage of EGFP+ (green) and EGFP- (blue) B-1a cells (B220 +CD19+ CD5+, determined by FACS) in the spleens and blood of lethally irradiated FVB recipient mice injected with Dnmt3a Δ/Δ bone marrow. P < 0.05 is indicated by (*), two-tailed Student’s t -test.

Article Snippet: Western blots were performed as previously described with use of the following antibodies: Dnmt3b (52A1018, Imgenex), Dnmt1 (H-300, Santa Cruz), and HDAC1 (ab7028, Abcam).

Techniques: Western Blot, Negative Control, Control, Expressing, Isolation, Irradiation, Injection, Staining, Two Tailed Test

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 1 Mahanine restores RASSF1A expression by demethylating its promoter and all three DNMTs control RASSF1A expression. A. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days. Methylation-specific PCR was performed to detect the methylated (M) and un-methylated (UM) status of RASSF1A promoter. B. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days, following which RASSF1A expression was assessed by RT-PCR. GAPDH was used as an internal control. C. PC3 cells were transfected with shRNA for DNMT1, DNMT3A, DNMT3B or scrambled shRNA. Forty-eight hours after transfection, cells were harvested for RT-PCR analyses to assess RASSF1A expression. GAPDH was used as an internal control. For DNMT3A, two shRNAs were used to confirm the result. D. BPH1 cells were transfected with expression vectors of DNMT1, DNMT3A, DNMT3B or empty vector control. Forty-eight hours after transfection cells were collected for RT-PCR analyses to determine RASSF1A, DNMT1, DNMT3A and DNMT3B expression levels. GAPDH was used as an internal control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Expressing, Control, Methylation, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Plasmid Preparation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 2 Mahanine specifically down-regulates DNMT1 and DNMT3B. A. DNMT1, DNMT3A and DNMT3B cellular localization was visualized by immunofluorescent staining. PC3 cells were treated with DMSO (as control) or mahanine (10 μM) for 24 hours, following which they were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 488-tagged secondary antibodies and counterstained with propidium iodide. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or mahanine (10 μM) for 24 hours are shown (right panel). B. Cytoplasmic and nuclear fractions were separated from PC3 cells treated with DMSO or 10 μM mahanine for 24 hours. The isolated fractions were subjected to Western blot analysis to assess DNMT expression. The fold change in the expression of the respective DNMTs as compared to the control is indicated at the bottom of each immunoblot. Nucleolin and β-actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. C. PC3 and LNCaP cells were treated as indicated with DMSO or mahanine following which cells were lysed and the extracts were subjected to Western blot analysis to detect DNMT1, DNMT3B and DNMT3A protein levels (left). Quantitative estimations of the relative levels of DNMT1, DNMT3A and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin (right). Columns, mean; bars, SEM. *p < 0.05, significantly different from control. D. PC3 and LNCaP cells were treated with DMSO or 10 and 20 μM mahanine, respectively for 24 hours. Subsequently, cells were harvested for RT-PCR analysis to measure DNMT1 and DNMT3B expression. GAPDH was used as an internal control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Staining, Control, Incubation, Fluorescence, Microscopy, Isolation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 3 Mahanine degrades DNMTs via the ubiquitin-proteasomal pathway. A. PC3 cells were treated with 10 μM mahanine and 20 μM Z-VAD-FMK for 24 hours after which cellular protein lysates were subjected to Western blot analysis to detect DNMT1 and DNMT3B protein levels, β-actin was used as a loading control. B. Chymotrypsin-like proteasomal activity was measured in PC3 cells treated as indicated with mahanine and MG132 for 24 hours. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO control. C. LNCaP and PC3 cells were treated with the indicated doses of mahanine for 24 hours with or without MG132 (5 μM). Cell lysates were analyzed for DNMT1 and DNMT3B expression by Western blot. β-actin was used as a loading control. D. PC3 cells were treated with MG132 (5 μM) in the absence and presence of mahanine (10 μM) for 24 hours. Cell lysates were subjected to immunoprecipitation (IP) of DNMT1 or DNMT3B and immunoblotted (IB) for poly-ubiquitin, DNMT1 and DNMT3B.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Activity Assay, Expressing, Immunoprecipitation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 4 Mahanine down-regulates pAkt levels and PI3K/Akt inhibitor wortmannin reduces DNMT1 and DNMT3B protein levels. A. PC3 and LNCaP cells were treated with mahanine as indicated. The cell lysates were subjected to Western blot analysis for phospho Akt (pAkt), total Akt. Β-actin was used as a loading control. B. PC3 and LNCaP cells were treated with wortmannin (1 μM) for 24 hours. Cell lysates were subjected to Western blot analysis to measure DNMT1, DNMT3B, pAkt and total Akt levels. β-actin was used as a loading control. The fold change in expression of the respective proteins compared to control is indicated below. C. PC3 cells were treated with DMSO or wortmannin (1 μM) for 24 hours. DNMT1 and DNMT3B cellular localization was visualized by immunofluorescent staining. After 24h of treatment, cells were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 594-tagged secondary antibodies and counterstained with DAPI. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or wortmannin (1 μM) for 24 hours are shown (right panel).

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Western Blot, Control, Expressing, Staining, Incubation, Fluorescence, Microscopy

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 5 Mahanine disrupts the interaction of pAkt with DNMT1 and DNMT3B and constitutively active Akt (CA-Akt) stabilizes their cellular levels in the presence of mahanine. A. and B. LNCaP cells were treated with MG132 with or without 20 μM mahanine for 24 hours and the cell homogenates were subjected to co-immunoprecipitation (IP) for DNMT1 or DNMT3B and immunoblotted (IB) for phosphor-serine (pSer), pAkt, total Akt, DNMT1 and DNMT3B. C. BPH1 cells were transfected with an empty vector or a constitutively active Akt (CA-Akt) expression vector for 24 hours then were treated with or without mahanine (10 μM) for another 24 hours. Levels of total Akt, pAkt, DNMT1 and DNMT3B proteins were measured through Western blots. β-actin was used as a loading control. Quantitative estimations of relative levels of DNMT1 and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO treated control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 6 Mahanine restores RASSF1A expression by degrading DNMTs via Akt. Prostate cancer cells express high levels of activated Akt, which phosphorylates and stabilizes DNMT1 and DNMT3B against proteasomal degradation. DNMTs enter the nucleus and methylate the promoter of RASSF1A gene to silence the expression of RASSF1A. Treatment of mahanine inhibits PDK1 and thereby prevents activation of Akt, which in turn compromises the stability of DNMTs, increases their ubiquitination and induces proteasomal degradation. In the absence of DNMT1 and DNMT3B, the RASSF1A promoter is demethylated and its expression is restored in prostate cancer cells. GF: Growth factor; RTK: Receptor tyrosine kinase; TFs: Transcription factors; P: Phosphorylated; M: Methylated; Ub: Ubiquitinated.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Methylation

Journal: PLoS ONE

Article Title: 5-Aza-2’-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats

doi: 10.1371/journal.pone.0179469

Figure Lengend Snippet: (A) Representative immunoblots and total protein image. Effects of alcohol exposure on the mRNA (B) and protein (C) expressions of DNMT1, DNMT3A and DNMT3B in the mPFC (n = 12/group). The data represent the mean ± SEM. *** p < 0.001, **** p < 0.0001 vs. water-exposure controls.

Article Snippet: The membranes were blocked with 5%(w/w) non-fat dried milk in Tris-buffered saline (TBS) (500 mM NaCl, 20 mM Tris-HCl pH 7.4) containing 0.05% Tween-20 for 1.5 h and incubated overnight with one of the following antibodies at 4°C: anti-DNMT1 polyclonal antibody (1:1000; Epigentek), anti-DNMT3A polyclonal antibody (1:400; Epigentek), anti-DNMT3B polyclonal antibody (1:500; Epigentek), anti-Ntf3 polyclonal antibody (1:1000; Abcam), anti-TrkC monoclonal antibody (1:1000, Cell Signaling Technology).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: 5-Aza-2’-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats

doi: 10.1371/journal.pone.0179469

Figure Lengend Snippet: Fig 5A shows representative immunoblots and total protein image. 5-Aza-dc decreased the protein level of DNMT1 (B), whereas it reversed the alcohol-induced overexpression of DNMT3A (C) and DNMT3B (D). 5-Aza-dc prevented the Ntf3 reduction induced by chronic alcohol exposure (E) but had no effect on TrkC (F). (G) Differential expression of mRNA for DNMTs, Ntf3 and TrkC. * p < 0.05, ** p < 0.01, *** p < 0.001, differences compared to the Water+DMSO group; # p < 0.05, ## p < 0.01, ### p < 0.01, differences compared to the other corresponding groups; n = 8/group.

Article Snippet: The membranes were blocked with 5%(w/w) non-fat dried milk in Tris-buffered saline (TBS) (500 mM NaCl, 20 mM Tris-HCl pH 7.4) containing 0.05% Tween-20 for 1.5 h and incubated overnight with one of the following antibodies at 4°C: anti-DNMT1 polyclonal antibody (1:1000; Epigentek), anti-DNMT3A polyclonal antibody (1:400; Epigentek), anti-DNMT3B polyclonal antibody (1:500; Epigentek), anti-Ntf3 polyclonal antibody (1:1000; Abcam), anti-TrkC monoclonal antibody (1:1000, Cell Signaling Technology).

Techniques: Western Blot, Over Expression, Quantitative Proteomics

Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: DNMT3B represses TAL1 expression by methylation modification. A UALCAN database to study the methylation level of TAL1 in LUAD and LUSC datasets. B Prediction of CpG islands in the promoter region of TAL1 by the MethPrimer database. C The correlation between TAL1 methylation levels and gene expression levels in LUAD or LUSC was predicted in the SMART database. D Correlation between DNMT3B expression and TAL1 methylation in LUAD and LUSC by Pearson’s analysis. E UALCAN database to study DNMT3B expression in LUAD and LUSC. F RT-qPCR analysis verified DNMT3B expression in NSCLC cells and BEAS-2B cells. G RT-qPCR verified the knockdown efficiency of DNMT3B. H RT-qPCR detected TAL1 expression in A549 and H460 cells. I Dual luciferase assay to detect TAL1 luciferase activity in A549 and H460 cells. J ChIP assay to validate DNMT3B enrichment at the promoter of TAL1 in A549 and H460 cells. K. qMSP assay to detect the methylation status of TAL1 in A549 and H460 cells. Comparisons were made using unpaired t-test, one-way, or two-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant

Article Snippet: A proportion of the DNA sample (2%) was collected as Input, and the lysates were incubated with the corresponding antibodies anti-DNMT3B (1:100, NBP2-59291, Novus Biologicals), anti-TAL1 (1:1000, GTX116020, GeneTex, Irvine, CA, USA), control antibody IgG (1:500, NB810-56910-10 mg, Novus Biologicals) and precipitated, and the DNA-protein immunocomplexes were collected using Pierce protein A/G-agarose beads and treated with RNase A and proteinase K. qPCR was performed using promoter-specific primers for TAL1 and PKM2.

Techniques: Expressing, Methylation, Modification, Gene Expression, Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay

Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: DNMT3B activates the glycolytic pathway by inhibiting TAL1 expression. A Colony formation assay to detect the proliferation of A549 and H460 cells after 14 days of culture. B EdU assay verified the proliferative ability of A549 and H460 cells. C , D The Transwell assay detected the migratory and invasive ability of A549 and H460 cells. E TUNEL assay detected apoptotic rate. F Cell metabolism assay detected changes in lactate levels. G WB detection of protein levels of glycolysis-related proteins GLUT1, LDHA, and PDK1. Comparisons were made among multiple groups using one-way or two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: A proportion of the DNA sample (2%) was collected as Input, and the lysates were incubated with the corresponding antibodies anti-DNMT3B (1:100, NBP2-59291, Novus Biologicals), anti-TAL1 (1:1000, GTX116020, GeneTex, Irvine, CA, USA), control antibody IgG (1:500, NB810-56910-10 mg, Novus Biologicals) and precipitated, and the DNA-protein immunocomplexes were collected using Pierce protein A/G-agarose beads and treated with RNase A and proteinase K. qPCR was performed using promoter-specific primers for TAL1 and PKM2.

Techniques: Expressing, Colony Assay, EdU Assay, Transwell Assay, TUNEL Assay

Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: DNMT3B affects NSCLC progression in vivo by regulating the TAL1/PKM2 axis. A Tumor images and tumor weight of mice. B The tumor volume of mice was measured periodically. C WB detection of DNMT3B, TAL1, and PKM2 protein expression in tumor tissues of mice. D IHC detection of glycolysis-related proteins (GLUT1, LDHA, and PDK1), Ki67, and Cleaved-Caspase3 in the tumors of mice. E TUNEL detection of apoptosis in tumor tissues. Animal experiments had five mice in each group. One-way or two-way ANOVA was used for single-factor comparisons among multiple groups. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant

Article Snippet: A proportion of the DNA sample (2%) was collected as Input, and the lysates were incubated with the corresponding antibodies anti-DNMT3B (1:100, NBP2-59291, Novus Biologicals), anti-TAL1 (1:1000, GTX116020, GeneTex, Irvine, CA, USA), control antibody IgG (1:500, NB810-56910-10 mg, Novus Biologicals) and precipitated, and the DNA-protein immunocomplexes were collected using Pierce protein A/G-agarose beads and treated with RNase A and proteinase K. qPCR was performed using promoter-specific primers for TAL1 and PKM2.

Techniques: In Vivo, Expressing, TUNEL Assay