RNAi of cyclin D2 and cyclin D3 differently impact viral replication depending on MDM type.

<p><b>(A)</b> Effective and specific knockdown of <i>CCND2</i> and <i>CCND3</i> expression by siRNA. Relative mRNA expression of <i>CCND2</i> (left panel) and <i>CCND3</i> (right panel) in M-CSF (white bars) and GM-CSF (black bars) macrophages. mRNA of the corresponding cyclin was measured by quantitative PCR and normalized to GAPDH expression. Data represents mean ± SD of 3 different donors and is normalized to Mock-transfected M-CSF macrophages. <b>(B)</b> Protein expression and SAMHD1 activation in cyclin D2 and cyclin D3 knockdown macrophages. Western blot showing cyclin protein expression, SAMHD1 expression and activation in siRNA-treated M-CSF and GM-CSF macrophages. SAMHD1 inactivation is reduced in siCCND3 (D3) M-CSF macrophages and increased in siCCND2 (D2) GM-CSF macrophages compared to the corresponding non-targeting siRNA (NT). Hsp90 was used as loading control. A representative donor is shown. <b>(C)</b> Evaluation of cell proliferation in cyclin D2 and cyclin D3 knockdown macrophages. Ki67 staining of siRNA-treated M-CSF (upper panels) and GM-CSF (lower panels) derived macrophages. Histograms from a representative donor are shown. <b>(D)</b> HIV-1 replication in siRNA-treated M-CSF or GM-CSF macrophages. Transfected MDM were infected with a VSV-pseudotyped, GFP-expressing HIV-1 and infection measured 72h later by flow cytometry. Data represent percentage replication relative to mock-transfected cells in M-CSF (white bars) or GM-CSF (black bars) macrophages. Mean ± SD of 3 different donors performed in triplicate is shown. * p<0.05; ** p<0.005; *** p<0.0005.