FIGURE 6.

Knockdown of Vav3 expression in BMMs by shRNAs resulted in reduced osteoclastogenesis. A, analysis of Vav3 knockdown (Vav3^KD) effects via immunoblot analysis. Puromycin-selected BMMs transduced with retroviral supernatants harboring Vav3^KD cassettes expressing Vav3 shRNA 1 (sh#1) and Vav3 shRNA 2 (sh#2) separately or in combination (sh#1+2) were cultured with M-CSF (150 ng/ml) for 2 days. The cell lysates were normalized for total protein content. Vav3^KD was visualized via anti-Vav3 (K-19) immunoblotting (IB). β-Actin was used as a loading control. mock, empty vector control. B, the effect of Vav3^KD on OC differentiation. BMMs were infected by Vav3^KD-retroviral supernatants harboring shRNAs 1 and 2, and the Vav3^KD-BMMs were further cultured with puromycin and M-CSF (150 ng/ml) for 2 days. Puromycin-selected Vav3^KD-BMMs were differentiated into OCs with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 4 days. The OCs were photographed (top panel, original magnification, ×50) after TRAP staining. The OCs were analyzed using the TRAP solution assay (bottom left panel). The number of TRAP⁺ MNCs (at least three nuclei) was counted (bottom right panel). mock, empty vector control. **, p < 0.01. C, the effect of Vav3^KD on resorption pit formation. Resorption pits were visualized (top panel, original magnification, ×50). The summarized data from the resorption pit assays are shown in the bottom panel. *, p < 0.05. D, the effect of Vav3^KD expression on the activation of osteoclastogenic factors. OCs were differentiated from Vav3^KD-BMMs with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for the indicated times and subsequently analyzed via IB with antibodies that recognized phosphorylated or total NFATc1 (7A6), c-Fos (D-1), and CREB (Ser¹³³). The cell lysates were normalized for total protein content. The level of Vav3 expression was detected via anti-Vav3 IB. The relative protein level was calculated after normalization to β-actin protein input (right panel). The relative level of phosphorylated CREB was calculated after normalization to total CREB protein input (right panel). β-Actin was used as the loading control. E, the effect of Vav3^KD on the expression of osteoclastogenic markers. Total RNA was prepared from Vav3^KD-OCs and subjected to real time PCR analysis. The data were normalized to β-actin. All experiments were performed at least three times with similar results.