METH use results in increased global DNA methylation in the brains of HIV seropositive cases.

<p><b><i>A.</i></b> Global methylation was determined by ELISA quantification of 5-mC on genomic DNA from frontal cortex samples. Significant increase in DNA methylation was observed in HIV seropositive METH users. *p<0.05 by Wilcoxon matched pairs test. <b><i>B.</i></b> Cellular HIV-1 RNA levels correlate with DNA methylation levels as determined by Spearman correlation between global DNA methylation and log HIV-1 RNA in the brain R = 0.527 with p = 0.01. <b><i>C.</i></b> Quantitative real-time PCR showed significant increase in DNMT1 transcript levels on HIV seropositive individuals who used METH. <b><i>D.</i></b> METH exposure did not alter mRNA levels of DNMT3B, a closely related family member reported to have redundant functions to DNMT1 in the brain. *p<0.05 by Wilcoxon matched pairs test. <b><i>E.</i></b> Western blot analysis of DNMT1 protein content in the nucleus showing representative HIV+METH− and HIV+METH+ cases. <b><i>F.</i></b> Image analysis showing integrated pixel intensity of Dnmt1 immunoreactivity. <b><i>G.</i></b> Immunofluorescence detection of DNMT1 on frontal cortex sections. Green fluorescent signal correspond to DNMT1 immunoreactivity and blue signal corresponds to DAPI nuclear staining. <b><i>H.</i></b> Image analysis showing average DNMT1 positive nuclear counts. Bar represents 10 µm. One way ANOVA was used to determine statistical significance, *p<0.05.