Journal: Molecular carcinogenesis

Article Title: miR-148a Dependent Apoptosis of Bladder Cancer Cells is Mediated in Part by the Epigenetic Modifier DNMT1

doi: 10.1002/mc.22319

Figure Lengend Snippet: A. Whole cell lysates from T24 cells treated either with miR-148a mimic or control oligonucleotide were used for western blot analysis of Bcl-2. Tubulin served as loading control. B. Whole cell lysates from SV-HUC-1, T24, TCCSUP, J82, and UM-UC-3 cells were used for western blot analysis of DNMT1. Actin served as loading control. C. Whole cell lysates from T24 and UM-UC-3 cells treated with either miR-148a mimic, DNMT1 targeting siRNA, or a control oligonucleotide were used for western blots for DNMT1. Tubulin or actin served as loading controls. D. Proliferation was assessed in T24 and UM-UC-3 cells treated with either DNMT1 targeting siRNA or a control oligonucleotide. ** denotes p-value ≤ 0.01, *** denotes p-value ≤ 0.001. E. qPCR was used to assess miR-148a expression in response to either control oligonucleotide (C) or DNMT1 targeting siRNA (D1) in both T24 (24) and UM-UC-3 (U3) cells.

Article Snippet: DNMT1-PCDNA3.1 was created by inserting DNMT1 sequence excised from an LZRS vector (LZRS-DNMT1 was a gift from Paul Khavari (Addgene plasmid # 24952)) into the multiple cloning site of PCDNA3.1.

Techniques: Western Blot, Expressing

Journal: Molecular carcinogenesis

Article Title: miR-148a Dependent Apoptosis of Bladder Cancer Cells is Mediated in Part by the Epigenetic Modifier DNMT1

doi: 10.1002/mc.22319

Figure Lengend Snippet: A. Cell cycle analysis by PI staining and flow cytometry was performed on DNMT1 siRNA treated or control oligonucleotide treated T24 cells and UM-UC-3 cells. B. Whole cell lysates from T24 and UM-UC-3 cells treated with either siRNA targeting DNMT1 or a control oligonucleotide were used for western blots for cleaved caspase-3 and cleaved PARP. Tubulin served as a loading control. C. Annexin-V staining coupled to flow cytometry was used to assess apoptosis on T24 and UM-UC-3 control oligonucleotide treated cells and DNMT1 targeting siRNA treated cells * denotes p-value ≤0.05, ** denotes p-value ≤ 0.01.

Article Snippet: DNMT1-PCDNA3.1 was created by inserting DNMT1 sequence excised from an LZRS vector (LZRS-DNMT1 was a gift from Paul Khavari (Addgene plasmid # 24952)) into the multiple cloning site of PCDNA3.1.

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Western Blot

Journal: Molecular carcinogenesis

Article Title: miR-148a Dependent Apoptosis of Bladder Cancer Cells is Mediated in Part by the Epigenetic Modifier DNMT1

doi: 10.1002/mc.22319

Figure Lengend Snippet: A. Whole cell lysates from DNMT1 overexpressing and control empty vector UM-UC-3 cells were used for western blots to demonstrate overexpression of DNMT1. Tubulin served as a loading control. B. Whole cell lysates from DNMT1 overexpressing and control empty vector UM-UC-3 cells treated either with control oligonucleotide or miR-148a mimic were used for western blots of PARP. Tubulin served as a loading control. C. DNMT1 overexpressing and control empty vector UM-UC-3 cells treated either with control oligonucleotide or miR-148a mimic were used for colony formation assays to demonstrate the effect of DNMT1 overexpression on miR-148a induced reduction of viability. PCDNA3.1 = control empty vector.

Article Snippet: DNMT1-PCDNA3.1 was created by inserting DNMT1 sequence excised from an LZRS vector (LZRS-DNMT1 was a gift from Paul Khavari (Addgene plasmid # 24952)) into the multiple cloning site of PCDNA3.1.

Techniques: Plasmid Preparation, Western Blot, Over Expression

Journal: Molecular carcinogenesis

Article Title: miR-148a Dependent Apoptosis of Bladder Cancer Cells is Mediated in Part by the Epigenetic Modifier DNMT1

doi: 10.1002/mc.22319

Figure Lengend Snippet: A. T24 and UM-UC-3 cells were transfected with either control oligonucleotide or miR-148a mimic and 24 hours post transfection were treated with either 0.5μM cisplatin or vehicle. Cell viability was assessed 48 hours post drug treatment using the CCK-8 reagent. B. T24 and UM-UC-3 cells were plated in 35mm dishes and were subjected to identical treatments as described in A. 48 hours post drug treatment, cells were stained with crystal violet and images were taken. C. T24 and UM-UC-3 cells were transfected with either control oligonucleotide or anti-miR-148a mimic and 24 hours post transfection were treated with either 0.5μM cisplatin or vehicle. Cell viability was assessed 48 hours post drug treatment using the CCK-8 reagent. D. T24 and UM-UC-3 cells were transfected with either control oligonucleotide or DNMT1 targeting siRNA and 24 hours post transfection were treated with either 0.5uM cisplatin or vehicle. Cell viability was assessed 48 hours post drug treatment using the CCK-8 reagent. * denotes p-value ≤ 0.05, ** denotes p-value ≤ 0.01, *** denotes p-value ≤ 0.001.

Article Snippet: DNMT1-PCDNA3.1 was created by inserting DNMT1 sequence excised from an LZRS vector (LZRS-DNMT1 was a gift from Paul Khavari (Addgene plasmid # 24952)) into the multiple cloning site of PCDNA3.1.

Techniques: Transfection, CCK-8 Assay, Staining

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial-mesenchymal transition and subcellular re-localization of Beta-Catenin.

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: Figure 1. (a). Schematic diagram of DNMT1 exons and protein domains of HCT116 and HCT116-Dnmt1Δ3−5 cells. Exon configuration of WT DNMT1, with annotation of primer sites used for analysis of DNMT1Δ3−5, and highlighted region of genomic deletion in this cell line. Protein domain schematic based on Pfam sites in Dnmt1 and Dnmt1Δ3−5. (b). Analysis of genomic Dnmt1 status in HCT116- Dnmt1Δ3−5 cells. Polymerase chain reaction analysis of Dnmt1 in HCT116-Dnmt1Δ3−5 cells in comparison to the endogenous WT gene in HCT116 cells. mRNA was extracted from cells, converted to cDNA by reverse transcription and cDNA analysed by PCR using primers specific to DNMT1 exons 1 and 6 (Table 2). PCR products were then analysed by gel electrophoresis resulting in a reduced DNMT1 product size in HCT116-Dnmt1Δ3−5 cells. (c). Western blot analysis of Dnmt1 and interaction partners in HCT116 and HCT116- Dnmt1Δ3−5 cells. Fifteen micrograms of whole protein lysates from HCT116 and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D59A4 antibody, see Table 2), Usp7, and Beta-Catenin. Tubulin protein abundance was used as a loading control. N = 3. (d). Nuclear-enriched protein samples of HCT116 and HCT116-Dnmt1Δ3−5 cells prepared for mass spectrometry analysis of their nuclear proteomic profile. Fifteen micrograms of nuclear- enriched protein lysates from HCT116 and HCT116-Dnmt1Δ3−5 cells, three replicates of each, were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D63A6 antibody, see Table 2). Lamin A/C protein abundance was used as a loading control for the nuclear-enriched lysate. These samples were then taken forward and prepared for nuclear proteomic analysis by data independent mass spectrometry analysis.

Article Snippet: HCT116Dnmt1Δ3−5 cells were transfected with pcDNA3 vector containing WT full length DNMT1 (36,939, AddGene) and empty pcDNA3 vector as a transfection control (10,792, AddGene).

Techniques: Polymerase Chain Reaction, Comparison, Reverse Transcription, Nucleic Acid Electrophoresis, Western Blot, Expressing, Quantitative Proteomics, Control, Mass Spectrometry

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial-mesenchymal transition and subcellular re-localization of Beta-Catenin.

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: Figure 3. (a). Analysis of nuclear enriched and whole cell protein lysates for protein abundance of EMT markers. Fifteen micrograms of nuclear-enriched or whole cell protein lysates from HCT116 and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Vimentin, N-Cadherin, and E-Cadherin. Tubulin protein abundance was used as a loading control for the whole cell protein lysate and Histone H3 protein abundance was used as a loading control for the nuclear-enriched lysate. N = 3. (b). Analysis of nuclear enriched and whole cell protein lysates for protein abundance of Delta-Catenin. Fifteen micrograms of nuclear-enriched or whole cell protein lysates from HCT116 and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Delta-Catenin. ß-Actin protein abundance was used as a loading control for the whole cell protein lysate and Histone H3 protein abundance was used as a loading control for the nuclear-enriched lysate. N = 3. (c). Corresponding mass spectrometry protein quantification of Dnmt1 and key EMT protein markers. (d). Western blot analysis of transcription-factors associated with EMT. Fifteen micrograms of nuclear- enriched protein lysates from HCT116 and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein abundance of Dnmt1 (D63A6 antibody, see Table 2), Zeb1, Slug, Snail, and Twist. Lamin A protein abundance was used as a loading control. N = 3.

Article Snippet: HCT116Dnmt1Δ3−5 cells were transfected with pcDNA3 vector containing WT full length DNMT1 (36,939, AddGene) and empty pcDNA3 vector as a transfection control (10,792, AddGene).

Techniques: Quantitative Proteomics, Western Blot, Expressing, Control, Mass Spectrometry

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial-mesenchymal transition and subcellular re-localization of Beta-Catenin.

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: Figure 4. (a). Analysis of nuclear and cytosolic enriched protein lysates for Dnmt1 and Beta-Catenin localization. Fifteen micrograms of nuclear-enriched or cytosol-enriched protein lysates from HCT116 and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D63A6 antibody, see Table 2) and Beta- Catenin. Lamin B1 protein abundance was used as a loading control for the nuclear-enriched lysate. N = 3. (b). Immunofluorescence analysis of Beta-Catenin and Dnmt1 localization in HCT116 and HCT116-Dnmt1Δ3−5 cells. Localization of Dnmt1 and Beta-Catenin protein was assessed using fluorophore-conjugated secondary antibodies and DAPI fixing agent. Channels are presented separately in addition to merged. Co-localization of Dnmt1 and Beta-Catenin was analysed using FIJI and MBF plugin, result of which is displayed in the ‘co-localization’ panel where areas of co-localization of Dnmt1 (red) and Beta-catenin (green) are highlighted in white.

Article Snippet: HCT116Dnmt1Δ3−5 cells were transfected with pcDNA3 vector containing WT full length DNMT1 (36,939, AddGene) and empty pcDNA3 vector as a transfection control (10,792, AddGene).

Techniques: Western Blot, Expressing, Quantitative Proteomics, Control, Immunofluorescence

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial-mesenchymal transition and subcellular re-localization of Beta-Catenin.

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: Figure 5. (a). Dnmt1 shRNA knockdown analysis on Vimentin protein abundance. Fifteen micrograms of whole cell protein lysates from shRNA knockdown HCT116 cells, HCT116-GFP controls cells and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D63A6 antibody, see Table 2) and Vimentin. A-Tubulin and Lamin A/C protein abundance were used as a loading control. N = 3. (b). Dnmt1 shRNA knockdown analysis on E-Cadherin protein abundance. Fifteen micrograms of whole cell protein lysates from shRNA knockdown HCT116 cells, HCT116-GFP controls cells and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D63A6 antibody, see Table 2) and E-Cadherin. A-Tubulin protein abundance was used as a loading control. N = 3. (c). Dnmt1 shRNA knockdown analysis on N-Cadherin protein abundance. Fifteen micrograms of whole cell protein lysates from shRNA knockdown HCT116 cells, HCT116-GFP controls cells and HCT116-Dnmt1Δ3−5 cells were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D63A6 antibody, see Table 2) and N-Cadherin. A-Tubulin protein abundance was used as a loading control. N = 3. (d). The effect of wild-type Dnmt1 protein expression rescue in HCT116-Dnmt1Δ3−5 cells on Vimentin protein abundance. Fifteen micrograms of whole cell protein lysates from HCT116 cells, HCT116-Dnmt1Δ3−5 cells, and HCT116-Dnmt1Δ3−5 cells transfected with either a full-length WT Dnmt1 expression vector or an empty control were loaded onto a single phase 8% SDS gene and analysed by western blot analysis for protein expression of Dnmt1 (D63A6 antibody, see Table 2). Tubulin protein abundance was used as a loading control. N = 2.

Article Snippet: HCT116Dnmt1Δ3−5 cells were transfected with pcDNA3 vector containing WT full length DNMT1 (36,939, AddGene) and empty pcDNA3 vector as a transfection control (10,792, AddGene).

Techniques: shRNA, Knockdown, Quantitative Proteomics, Western Blot, Expressing, Control, Transfection, Plasmid Preparation

Journal: Science translational medicine

Article Title: Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer

doi: 10.1126/scitranslmed.adf6732

Figure Lengend Snippet: (A) DNMT1, DNMT3A, and DNMT3B mRNA expression in benign prostate (n = 29), PCA (n = 75), CRPC-Adeno (n = 73), and NEPC (n = 36). Two-tailed t test was used. FPKM, fragments per kilobase of exon per million mapped reads. (B) DNMT1, DNMT3A, and DNMT3B mRNA expression in NEPC. Two-tailed t test was used. (C) A survey of DNMT mRNA expression across six prostate cell lines, organoids, and PDX models. (D) Correlation of DNMT expression and NEPC score in the International SU2C/PCF Dream Team dataset (11). (E) Representative IHC images of PCA, CRPC-Adeno, and NEPC stained by hematoxylin and eosin (H&E) and with CHGA, AR, DNMT1, DNMT3A, and DNMT3B antibodies. Scale bars, 100 μm. *P < 0.05, **P < 0.01, ****P < 0.0001. TPM, transcripts per million.

Article Snippet: Cells were transfected with nontargeting control pool or SMARTpool siRNAs (Horizon) to knock down DNMT1 and B7-H3 using electroporation (4D-Nucleofector Core Unit, Lonza). pcDNA3 vectors were used to overexpress DNMT1 [pcDNA3/Myc-DNMT1 was a gift from A. Riggs (Addgene, plasmid #36939; http://n2t.net/addgene:36939 ; RRID:Addgene_36939)] and DNMT3A [pcDNA3/Myc-DNMT3A was a gift from A. Riggs (Addgene, plasmid #35521; http://n2t.net/addgene:35521 ; RRID: Addgene_35521)] by an electroporation method ( 60 , 61 ).

Techniques: Expressing, Two Tailed Test, Staining

Journal: Science translational medicine

Article Title: Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer

doi: 10.1126/scitranslmed.adf6732

Figure Lengend Snippet: (A) DNMT1/DNMT3A protein expression after DNMT1/3A knockout in WCM154 (NEPC) cells. GAPDH, glyceraldehyde phosphate dehydrogenase. (B) Cell viability analyses in WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. A two-way ANOVA test was used. (C) Subcutaneous injection of mice with 1 × 107 cells and tumor appearance ratio in WCM154 sgGFP, sgDNMT1, and sgDNMT3A. (D) Postinjection time and tumor volumes in WCM154 sgGFP, sgDNMT1, and sgDNMT3A. A two-way ANOVA test was used. (E) Representative IHC images of WCM154 sgGFP, sgDNMT1, and sgDNMT3A tumors stained by H&E. Scale bars, 100 μm. (F) Representative IHC images of WCM154 sgGFP, sgDNMT1, and sgDNMT3A tumors stained by DNMT1 and DNMT3A. Scale bars, 100 μm. (G) Orthotopic injection in murine prostate with 2 × 105 cells and tumor appearance and liver metastasis ratio in WCM154 sgGFP, sgDNMT1, and sgDNMT3A. (H) Representative images of murine prostate injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. (I) Weights of gGU system injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. A two-tailed t test was used. (J) Representative IHC images of murine prostate injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells stained by H&E. Scale bars, 100 μm. (K) Representative IHC images of murine prostate injected with WCM154 sgGFP and sgDNMT3A cells stained by DNMT3A. Scale bars, 100 μm. (L) Multiple liver metastatic lesions generated from murine prostate injected with WCM154 sgGFP cells (left). Representative IHC images of liver metastatic tumors stained by H&E, DNMT1, DNMT3A, CHGA, and SYP. Scale bars, 100 μm. **P < 0.01, ****P < 0.0001.

Article Snippet: Cells were transfected with nontargeting control pool or SMARTpool siRNAs (Horizon) to knock down DNMT1 and B7-H3 using electroporation (4D-Nucleofector Core Unit, Lonza). pcDNA3 vectors were used to overexpress DNMT1 [pcDNA3/Myc-DNMT1 was a gift from A. Riggs (Addgene, plasmid #36939; http://n2t.net/addgene:36939 ; RRID:Addgene_36939)] and DNMT3A [pcDNA3/Myc-DNMT3A was a gift from A. Riggs (Addgene, plasmid #35521; http://n2t.net/addgene:35521 ; RRID: Addgene_35521)] by an electroporation method ( 60 , 61 ).

Techniques: Expressing, Knock-Out, Injection, Staining, Two Tailed Test, Generated

Journal: Science translational medicine

Article Title: Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer

doi: 10.1126/scitranslmed.adf6732

Figure Lengend Snippet: (A) Heatmap of DNMT mRNA gene expression, NEPC markers, apoptosis marker, and cell proliferation markers in WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. (B) GO enrichment analysis in down-regulated genes after DNMT1 knockout. WT, wild type; MAPK, mitogen-activated protein kinase. (C) Gene set enrichment analysis in down-regulated genes after DNMT3A knockout. NES, normalized enrichment score. (D) WB analyses of NE markers in WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. (E) Representative IHC images of subcutaneous tumors injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A stained by H&E, CHGA, SYP, and INSM1. Scale bars, 200 μm. (F) WB analyses of NE markers after overexpression of DNMT1/3A in DNMT1/3A knockout cells. (G) Cell viability analyses in WCM154 si_Control and si_DNMT1. A two-tailed t test was used. (H and I) WB analyses of NE markers after DNMT1 silencing in WCM154 and WCM155 cells. ****P < 0.0001.

Article Snippet: Cells were transfected with nontargeting control pool or SMARTpool siRNAs (Horizon) to knock down DNMT1 and B7-H3 using electroporation (4D-Nucleofector Core Unit, Lonza). pcDNA3 vectors were used to overexpress DNMT1 [pcDNA3/Myc-DNMT1 was a gift from A. Riggs (Addgene, plasmid #36939; http://n2t.net/addgene:36939 ; RRID:Addgene_36939)] and DNMT3A [pcDNA3/Myc-DNMT3A was a gift from A. Riggs (Addgene, plasmid #35521; http://n2t.net/addgene:35521 ; RRID: Addgene_35521)] by an electroporation method ( 60 , 61 ).

Techniques: Expressing, Marker, Knock-Out, Injection, Staining, Over Expression, Two Tailed Test