Figure 4

Effect of R5 HIV-1 infection on osteoclast differentiation. (A) CD14⁺ monocytes were cultured for 6 days with M-CSF plus RANKL. The cells were infected with JR-FL (2.5 × 10⁵ IFU/ml) in the absence or presence of 4 μg/ml TFV or with AT-2-inactivated JR-FL (2.5 × 10⁵ IFU/ml). Five days after infection, the cells were fixed and stained for TRAP. Scale bar, 300 μm. (B) CD14⁺ monocytes were cultured for 6 days with M-CSF plus RANKL (OC) or M-CSF alone (Mph). The cells were infected with JR-FL or with AT-2-inactivated JR-FL at the indicated doses. Five days after infection, total RNA was prepared and the expressions of the osteoclast markers, such as ACP5/TRAP, CTSK, and CALCR, were analyzed by quantitative real-time RT-PCR. (C) CD14⁺ monocytes were cultured for 6 days with M-CSF plus RANKL (OC) or M-CSF alone (Mph). The osteoclasts were infected with JR-FL in the presence of TFV at the indicated doses. Five days after infection, the expressions of ACP5/TRAP, CTSK, and CALCR were analyzed by quantitative real-time RT-PCR. (B, C) The relative mRNA expression level of each marker is shown as fold induction in comparison to the expression in the uninfected macrophages. The data were standardized by the level of β-actin expression in each sample. (*p < 0.05, **p < 0.01 by t test).