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: 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