Fig. 5

5’ LTR DNA methylation is resistant to HDAC depletion in the HIV-1 latency model cell line. a HDAC1 relative mRNA levels at days 0 (control), 3, and 6 after siCTRL or siHDAC1 transfection in the 2D12 cell line as determined by qRT-PCR. 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 HDAC2 relative mRNA levels at days 0 (control), 3, and 6 after siCTRL or siHDAC2 transfection in the 2D12 cell line determined by qRT-PCR. 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. c Latent HIV-1 provirus reactivation levels after 6 days of HDAC2 and HDCA1 knockdown. 2D12 cells that were transfected with siCTRL or with siHDAC2 and siHDAC1 are shown, either without cellular stimulation (left column) or after 24 h of TNF-α and PMA stimulation (right column). Three FACS analyses of three biological replicates are overlaid; the mean percentage of EGFP-positive cells is shown on the x-axis, whereas the mean fluorescence intensity of EGFP is shown on the y-axis. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. d CpG methylation profile of 5’ LTR in the non-stimulated 2D12 cell line. Latent HIV-1 provirus CpG methylation levels at the 5’ LTR sequences after 6 days of HDAC2 knockdown in the 2D12 cell line. 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