Journal: Cancers

Article Title: Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation

doi: 10.3390/cancers12020447

Figure Lengend Snippet: ( A ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to the indicated concentrations of 2b or 4c . Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as loading control. Blots are representative of two independent experiments. Right: Densitometric analysis of protein levels is reported. ( B ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to 4c at 1 µM and co-treated with bortezomib (when indicated) used at 10 nM. Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as a loading control. Blots are representative of three independent experiments. Right: Densitometric analysis of protein levels is reported. Data are represented as mean ± SEM. Significance is represented as * p < 0.05 related to the control.

Article Snippet: The following primary antibodies were used for immunoblotting: α-DNMT1 (Novus Biologicals, Littleton, CO, USA). α-DNMT3a (SantaCruz Bio-technologies, CA, USA) and α-GAPDH (Millipore Corp., Bedford, MA USA), used as a loading control.

Techniques: Western Blot, Expressing, Control

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: DNMT1 target genes. ( A ) Venn diagram depicts numbers of genes upregulated in clones containing empty vector pEF1αIRESpuro (E1, E2, E3) of HCT116 DNMT1 − / − hypomorphs compared to parental HCT116. ‘ DNMT1 −/− ’ is the parental DNMT1 −/− hypomorph clone. A total of 229 genes were found upregulated 1.4-fold or greater in all four DNMT1 −/− clones. From the 229 gene list, genes upregulated 1.4-fold also in DNMT3b −/− were eliminated and yielded 135 genes. ( B ) RT–PCR validation for upregulation of genes in DNMT1 −/− (E1) cells. Fold change relative to HCT116 is calculated. cMYC was used as a negative control for a gene with unchanged expression in the DNMT1 −/− cells. Microarray fold changes for individual genes are written below. ( C ) Western blot to assess transient knockdown of DNMT1 in HCT116 cells transfected with DNMT1 siRNA (DNMT1si) or non-target siRNA (NTsi) for 72 h. αβactin serves as a loading control. ( D ) RT–PCR analysis of DSCR8 , DTX3 , MAGEA10 , TXNIP and cMYC genes in HCT116 cells transfected with non-target control siRNA (dark grey bar) or DNMT1 siRNA (light gray bar). Fold change relative to non-target control was calculated. Bars = standard error of three independent experiments. ( E ) ChIP at DSCR8 , DTX3 , MAGEA10 , TXNIP and cMYC promoter regions for αDNMT1. Samples included HCT116 and DNMT1 −/− (E1) cell lines. RT–PCR was conducted and the average levels of enrichment relative to input and standard errors were calculated for three independent ChIP experiments.

Article Snippet: Antibodies utilized in western blot analysis were as follows: αDNMT1 (Sigma D4567) 1:2000, αDNMT1 (epitomics 2788-1) 1:3000, αβactin (Sigma 5441) 1:10 000, αLSD1 (Millipore 09-058) 1:10 000, αCBP (Santa Cruz sc-369) 1:1000, αLaminB (Santa Cruz) 1:2000, α-tubulin (Sigma T6074) 1:10 000 and αGAPDH (Millipore) 1:10 000.

Techniques: Clone Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Expressing, Microarray, Western Blot, Transfection

Journal:

Article Title: Characterization of Dnmt3b:thymine-DNA glycosylase interaction and stimulation of thymine glycosylase-mediated repair by DNA methyltransferase(s) and RNA

doi: 10.1016/j.jmb.2008.02.049

Figure Lengend Snippet: DNA methyltransferases stimulate base excision repair of T·G mismatches. A: Design and testing of oligodeoxyribonucleotides (ODNs) used in a mismatch repair assay. (a) Sequence of the double-stranded ODN. The sense strand contains either a normal CCGG site or a mutated CTGG site. The antisense strand contains either C or 5mC within an MspI/HpaII recognition site. Positions of variable nucleotides y and x are shown in bold. The MspI/HpaII recognition sequence is indicated with a bar. The asterisk indicates the position of the 32P label. (b) Predicted sensitivity to MspI or HpaII digestion of the control C·GC and C·GmC ODNs compared to the mismatched T·GC and T·GmC ODNs. (c) MspI digestion of the control ODNs results in the expected 26 nt long radiolabeled fragment from both controls, while a 26 nt long fragment is only observed after HpaII digestion of the unmethylated control. In contrast, T·G mismatch ODNs are refractory to digestion by either enzyme. M, MspI. H, HpaII. B: De novo DNA methyltrasnferases stimulate repair of T·G mismatches. (a) Evaluation of Dnmt and Tdg protein levels in J1 and Dnmt null ES cells. Nuclear extracts prepared from each cell line were immunoblotted using antibodies specific for Dnmt1, Dnmt3a, Dnmt3b and Tdg to verify the expression of Tdg and the absence of specific Dnmt expression in each cell line relative to wild-type J1. Immunoblot for Pcna served as a loading control. (b) Predicted T·GC ODN sensitivity to digestion by MspI or HpaII depending on extent of repair/DNA methylation. (c) Typical results from one of three independent experiments in which the T·GC ODN was incubated with wild-type J1 or Dnmt null ES cell nuclear extracts followed by digestion with either MspI or HpaII. The expected 26 nt repair product produced as a result of digestion is indicated by the arrowhead. In the absence of either Dnmt3b and/or Dnmt3a there is a reduction in repair efficiency when compared to wild-type nuclear extracts (refer to text for discussion of Dnmt1 null extracts). As a control (last lane), T·GC ODN was used in a repair assay but was not digested. Lack of a 26 nt fragment indicates the ODN is being repaired and not merely nicked 5′ to the mismatched thymine. (d) Quantitation of differences in the extent of mismatch repair by extracts from normal ES cells (J1) and ES cells nullizygous for the indicated Dnmts. Error bars represent the mean (± SD) of three independent experiments. [*] P < 0.005

Article Snippet: Protein was transferred to PVDF membrane and analyzed by standard immunoblotting with protein specific antibodies: α-Dnmt1 (Santa Cruz, K-18), α-Dnmt3a (Imgenex), α-Dnmt3b (Imgenex), α-Tdg( 37 ), α-PCNA (Santa Cruz, sc-56) with appropriate secondary antibodies conjugated to horse radish peroxidase (HRP).

Techniques: Sequencing, Expressing, Western Blot, DNA Methylation Assay, Incubation, Produced, Quantitation Assay