Journal: International immunopharmacology

Article Title: The epigenetic-modified downregulation of LOXL1 protein mediates EMT in bladder epithelial cells exposed to benzo[a]pyrene and its metabolite BPDE.

doi: 10.1016/j.intimp.2024.113232

Figure Lengend Snippet: Fig. 5. DNMT3a and DNMT3b are key factors in the regulation of LOXL1 methylation modification. (A-D) The protein level of DNMT3a/3b was detected by WB assay in SV-HUC-1 cells exposed to the indicated concentrations (1 μM and 2 μM) of B[a]P/BPDE for 24 h (Fig. 5A), 1 month (Fig. 5B), 3 months (Fig. 5C), and 6 months (Fig. 5D); (E) DNMT3a knockdown construct was stably transfected into SV-HUC-1 cells, and knockdown efficiency was then determined using Western blot; (F) The efficiency of DNMT3a knockdown on downstream LOXL1 protein level was assessed using Western blot; (G) Stable transfection of DNMT3b knockdown plasmid into SV-HUC-1 cells and WB assay to detect its knockdown efficiency; (H) Western blot was used to detect the effect of DNMT3b knockdown on downstream protein LOXL1 expression.

Article Snippet: Antibodies specific against DNMT3a (sc-373905), DNMT3b (sc376043), LOXL1 (sc-166632), SLUG (sc-166476) were purchased from Santa Cruz Biotechnology (TX, USA); Antibodies specific against ECadherin (60335-1-Ig) and β-Actin (66009-1-Ig), were purchased from Proteintech (Wuhan, China).

Techniques: Methylation, Modification, Knockdown, Construct, Stable Transfection, Transfection, Western Blot, Plasmid Preparation, Expressing

Journal: International immunopharmacology

Article Title: The epigenetic-modified downregulation of LOXL1 protein mediates EMT in bladder epithelial cells exposed to benzo[a]pyrene and its metabolite BPDE.

doi: 10.1016/j.intimp.2024.113232

Figure Lengend Snippet: Fig. 6. B[a]P/BPDE exposure induces DNMT3a and DNMT3b expression in SV-HUC-1 cells by generating ROS. (A) Intracellular ROS levels were assessed using the DCFH-DA probe; (B) Quantitative analysis of fluorescence intensity; (C) Detection of intracellular ROS levels after NAC (3 mM) treatment with the ROS inhibitor using DCFH-DA probe; (D) Quantitative analysis of fluorescence intensity; (E) WB assay was performed to detect the impact of inhibitor NAC on down stream proteins expression in SV-HUC-1 cells; (F) Cell scratch assay was used to detect the impact of ROS inhibitor NAC on the migration ability of SV-HUC-1 cells.

Article Snippet: Antibodies specific against DNMT3a (sc-373905), DNMT3b (sc376043), LOXL1 (sc-166632), SLUG (sc-166476) were purchased from Santa Cruz Biotechnology (TX, USA); Antibodies specific against ECadherin (60335-1-Ig) and β-Actin (66009-1-Ig), were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Fluorescence, Wound Healing Assay, Migration

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: bioRxiv

Article Title: De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG islands

doi: 10.1101/676346

Figure Lengend Snippet: a) Many CGIs gain methylation upon expression of DNMT3B in DKO cells. Density scatter plot comparing mean CGI methylation levels in DKO cells and DKO cells expressing DNMT3B (DKO + DNMT3B). Green individual points denote CGIs significantly gaining methylation upon DNMT3B expression (≥20% methylation gain and Benjamini-Hochberg corrected Fisher’s exact tests p < 0.05). b) DNMT3B target CGIs are enriched in H3K36me3 marked regions. Barplot of the fold-enrichment observed for HCT116 histone modification peaks in the set of CGIs that significantly gain methylation when DNMT3B is expressed in DKO cells compared to the background of all CGIs observed in the experiment. P-values are from Fisher’s exact tests (1-sided for enrichment). c) Examples of DNMT3B target CGIs. Genome browser plots showing DNA methylation levels and HCT116 H3K36me3 ChIP signal. CGIs and genes are shown below the plots. CGIs gaining methylation when DNMT3B is expressed in DKO cells are indicated in light blue. +3B = DKO + DNMT3B; +3Bcd = DKO + catalytically dead DNMT3B. Scale for methylation data is 0 to 100%. For H3K36me3 it is 0 to 9.5. d) CGIs with the highest levels of H3K36me3 gain the greatest levels of DNA methylation. Boxplot showing gain of DNA methylation at CGIs when DNMT3B is expressed in DKO cells relative to their level of H3K36me3. CGIs were ranked by H3K36me3 and split into 10 equally sized groups. Lines=median; Box=25th–75th percentile; whiskers=1.5× interquartile range from box. e) DNMT3B localises to CGIs that gain methylation upon DNMT3B expression in DKO cells. qPCR analysis of CGIs following anti-T7 ChIP from T7-DNMT3B cells and HCT116 cells lacking the tag (T7-mock). Shown are the mean of 3 biological replicates with the standard deviation indicated by the error bars. P-values are from T-tests.

Article Snippet: To create the piggyBac DNMT3B expression vector, the DNMT3B2 sequence from pcDNA3/Myc-DNMT3B2 plasmid (Addgene plasmid 36942, a gift from A. Riggs) was subcloned using XbaI and BamHI into the pCG plasmid (a gift from N. Gilbert) downstream of the T7 tag.

Techniques: Methylation, Expressing, Modification, DNA Methylation Assay, Standard Deviation