Expression level analysis of DNMT1, 3A, 3B and G9a mRNA in mock vector and OAZ transfectants by qRT-PCR.

<p>Expression level of these mRNA was not altered by OAZ. The PCR result was normalized using the -beta-actin signal. Signal of the control (mock vector transfectant without ZnSO₄ treatment) was considered as 100 and other signals were expressed as relative value.</p

Intracellular level of polyamines, L-ornithine, SAM, dcSAM, SAH.

<p>Polyamine pool was depleted (A) but dcSAM and SAH, which were potential inhibitors of methylation reactions, increased (B) in the OAZ transfectant treated with ZnSO₄. Cellular level of SAM was constant through the experiments (B).</p

Quantification of global DNA methylation in the parental UM1 cells, mock vector and OAZ transfectants.

<p>Global methylation level of 5′-methyl-CMP (dm5′dCMP) and 5′dCMP in genome DNA of the parental UM1 cells, mock vector and OAZ transfectant was measured by scintillation counting. ³²P-labeled dm⁵CMP and CMP were produced by cleavage of the respective genome DNAs with the restriction enzymes <i>Msp</i>I and its methylation sensitive isoschizomer <i>Hpa</i>II and they were separated on a thin layer chromatography.</p

Metabolic pathway coupling between polyamine synthesis and methylation reactions.

<p>ODC is a key enzyme of polyamine synthesis by catalyzing from L-ornithine to putrescine. OAZ is an inhibitory molecule of ODC activity by mediating degradation of ODC protein. SAM serves as a methyl donor in methylation reactions as well as precursor of dcSAM production. dcSAM is an aminopropyl donor of polyamine biosynthesis but it also acts as a competitive inhibitor of SAM virtually in all methylation reactions. Inhibition of ODC activity leads to polyamine depletion and dcSAM accumulation, which inhibits methylation reactions.</p

ODC enzymatic activity assay in mock vector and OAZ transfectants.

<p>ODC activity (picomoles CO₂ per hour per milligram protein) of the parental UM1, the mock vector transfectant and OAZ transfectant with and without ZnSO₄ treatment was measured. Triplicate quantification was performed. Corrected ODC activity values were obtained by subtracting the value for blank. ODC activity of OAZ transfectant without ZnSO₄ treatment was suppressed because trace amount of ZnSO₄ in culture medium caused leakage of OAZ gene expression.</p

List of PCR primers for qRT-PCR.

<p>List of PCR primers for qRT-PCR.</p

DNMT enzymatic activity assay in mock vector and OAZ transfectants.

<p>Total DNMT activity assay was performed. DNMT activity was decreased by ∼65% in the OAZ transfectant treated with ZnSO₄. This result is correlated with the western blot result of DNMT3B.</p

Western immunoblot analysis of DNMTs proteins in mock vector and OAZ transfectants.

<p>Nuclear extracts were immunoblotted with anti-DNMTs antibodies. Protein level of DNMT1 (183 kDa) and DNMT3A (101 kDa) was constant but protein level of DNMT3B (110 kDa) was significantly decreased in the OAZ transfectant treated with ZnSO₄. The fain bands appeared in the DNMT3A and DNMT3B blots are isoforms of DNMT3A and 3B. Oct-1 (89 kDa) was used to monitor equal amount of protein loading in each lane.</p

Western immunoblot analysis of G9a protein in mock vector and OAZ transfectants.

<p>Nuclear extracts were immunoblotted with anti-G9a antibody. Expression of G9a protein (140 kDa) was decreased in the OAZ transfectant treated with ZnSO₄.</p

Continuity of each type of mass with the gubernaculum tract.

<p>Continuity of each type of mass with the gubernaculum tract.</p