Fig 3. RNAi of cyclin D2 and cyclin D3 differently impact viral replication depending on MDM type.

(A) Effective and specific knockdown of CCND2 and CCND3 expression by siRNA. Relative mRNA expression of CCND2 (left panel) and CCND3 (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) 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. (C) 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. (D) 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.