Hypermethylation of 5’ LTR DNA in the HIV-1 latency model cell line 2D12 is not maintainted by DNMT3B. a DNMT3B protein expression in 2D12 cell lines that were transfected with siCTRL and with siDNMT3B at days 3 and 6 after transfection as determined by the specific antibody is depicted on the left panel (upper band). Equal protein loading was verified by anti-γ-tubulin antibodies as depicted on the left panel (lower band). The DNMT3B relative mRNA levels at days 0 (control), 3, and 6 of siCTRL or siDNMT3B transfection in the 2D12 cell line as determined by qRT-PCR are shown in the right panel. mRNA expression was normalized to the expression of the RNA polymerase II, polypeptide A (POLR2A) housekeeping gene. The data are presented as the mean ± SD of technical triplicates. Three independent experiments were performed, representative example is shown. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. b Latent HIV-1 provirus reactivation levels after 6 days of DNMT3B knockdown. 2D12 cells were transfected with siCTRL or with siDNMT3B. The left column of histograms represents analyses without cellular stimulation whereas the right column represents histograms after 24 h of TNF-α and PMA stimulation. Three FACS analyses of three independent experiments are overlaid; the mean percentage of EGFP-positive cells is shown above the bar. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. c CpG methylation profile of 5’ LTR in non-stimulated 2D12 cells. The 5’ LTR methylation profiles were obtained from cells transfected with siCTRL or those transfected for 3 and 6 days with siDNMT3B. Methylation levels are presented as a mean percentage of methylated CpGs (mCpGs) in HIV-1 promoters. An analysis of promoter molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced molecule of the 5’ LTR. Mixture of genomic DNA isolated from three independent experiments was used for bisulfite analysis. The rectangle schematically represents the 5’ LTR regions U3, R, and U5 with individually analyzed CpG dinucleotides and transcription factor binding sites.