Figure 1.

The VDR is epigenetically repressed during HCMV infection. (A) Immunoblot showing VDR and GAPDH expression in HFF treated with DMSO (−) or 1 μM MG132 (+) proteasome inhibitor in mock- or CMV-infected cells. Ubiquitinated proteins are shown as a control for proteasome inhibitor effectiveness. Representative blot (left) and bar graph from multiple experiments (right panel). (B) RT-qPCR analysis of VDR mRNA expression in mock- or CMV-infected HFF treated with 50 μg/mL cycloheximide (chx, n = 2) starting at 6 h p.i. and harvested 24 h p.i. Asterisks indicate significant differences to the mock control sample (unpaired Student’s t test). (C) RT-qPCR analysis of VDR and intron-containing unspliced pre-mRNA (VDR-pre) expression in mock- and HCMV-infected HFF harvested 24 h p.i. Asterisks indicate significant differences to the mock control sample using unpaired Student’s t tests. (D) Immunoblots showing VDR expression compared to GAPDH loading control in HFF infected with CMV and treated with DMSO (vehicle), 1 μM TSA, or a combination of TSA and 10 nM calcitriol (VD3), harvested 24 h p.i. A representative blot is shown on the left, the right panel shows VDR quantification from multiple experiments with significant differences to the CMV DMSO control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. (E) Purified chromatin from mock- and CMV-infected HFF was immunoprecipitated with an anti-H3K27ac polyclonal antibody or normal goat serum (ctrl IP). The left panel shows qPCR results from three independent experiments. On the right, VDR promoter amplicons from a Taq-PCR were separated on an agarose gel. NTC = no template control.