expression vector pcdna3 myc dnmt1  (Addgene inc)

Journal: Cancer research

Article Title: GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

doi: 10.1158/0008-5472.CAN-16-2289

Figure Lengend Snippet: Altered Tumor Cell GAD1 Promoter Methylation and DNMT1 Expression Induced by Brain Microenvironment. A, UCSC Genome Browser plot of ENCODE data track of CpG Islands located around the GAD1 promoter. B, Promoter methylation-specific PCR (MSP) assay detecting methylation status in human GAD1 promoter region in vitro. C, (left) Bisulfite sequencing of CpG island 122 located in human GAD1 promoter region, (right) Percentage of Methylated CpG sites in sequenced region. D, MSP assay detecting methylation status in the human GAD1 promoter region in vivo. E, Bioinformatics analysis of GSE19184 showing normalized DNMT1 probe intensity in primary tumors or brain metastases arising from indicated cell lines. F, qRT-PCR of DNMT1 mRNA levels in primary tumors or brain metastases arising from MDA-MB231.

Article Snippet: Lentiviral-based expression vectors pcDNA3/Myc-DNMT1 (36939), pcDNA3.1-Peredox-mCherry (32383) and packaging vectors were purchased from Addgene.

Techniques: Methylation, Expressing, MSP Assay, In Vitro, Methylation Sequencing, In Vivo, Quantitative RT-PCR

Journal: Cancer research

Article Title: GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

doi: 10.1158/0008-5472.CAN-16-2289

Figure Lengend Snippet: Brain Microenvironment-Induced Down-Regulation of DNMT1 Reactivates GAD1 Expression. A, qRT-PCR of DNMT1 mRNA expression after 48 hours co-culture with either CAF or glia cells. (left) MDA-MB-231; (right) A375SM. B, qRT-PCR of DNMT1 mRNA expression of MDA-MB-231 cultured either with 100% conditioned media from either CAF or glia cells or 50% mix of conditioned media and fresh media. C, Cytokine screen of glia and CAF conditioned media. (left) MA plot of Log [mean expression of Glia/CAF] of 73 cytokines analyzed. *: differentially expressed cytokines (adjusted p < 0.1) (right) Heatmap of differentially expressed cytokines. D, Network analysis of differentially expressed cytokines. E, Impact of extracellular clusterin on DNMT1 and GAD1 expression. (left) Cytokine expression profile of clusterin in conditioned media from CAFs or glia cells. (right) qPCR of GAD1 and DNMT1 mRNA expression in MDA-MB-231 cells treated with control or 200 ng of clusterin. F, qRT-PCR of GAD1 and DNMT1 mRNA expression in tumor cells genetic knockdown of glia derived-clusterin. (left) qRT-PCR of clusterin mRNA expression in glia cells. (right) qRT-PCR of GAD1 and DNMT1 mRNA expression in MDA-MB-231 cells co-cultured with control glia or siClusterin glia cells. G, qRT-PCR of mRNA levels in tumor cells after 48 hours co-culture with primary glia cells. Prior to co-culture, tumor cells were transfected with either vector control or DNMT1 over expression plasmid for 24 hours. (left) DNMT1 mRNA expression; (right) GAD1 mRNA expression under glia co-culture. H, Proliferation of MDA-MB-231 cells after DNMT1 overexpression and co-culture with glia cells for 48 hours.

Article Snippet: Lentiviral-based expression vectors pcDNA3/Myc-DNMT1 (36939), pcDNA3.1-Peredox-mCherry (32383) and packaging vectors were purchased from Addgene.

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Cell Culture, Derivative Assay, Transfection, Plasmid Preparation, Over Expression