Fig. 4: Pld2 deficiency accelerates the migration of osteoclast lineage cells by activating M-CSF-induced Akt signaling.

a WT and Pld2^−/− BMMs were cultured with M-CSF at the indicated concentrations. After 3 days, BrdU incorporation was measured. b WT and Pld2^−/− BMMs were cultured with M-CSF (30 ng/ml). The extent of apoptosis was measured using cell death ELISA. c, d Cytokine-starved BMMs (c) or pre-OCs (d) from WT and Pld2^−/− mice were subjected to migration assays after treatment with M-CSF. Representative images of migrated cells stained with crystal violet (left) and percentage of migrated cells (right). e, f Cytokine-starved WT and Pld2^−/− BMMs were stimulated with M-CSF (50 ng/ml) for the indicated times. Immunoblotting was used to assess the phosphorylation of Akt and ERK (e) and GSK3β (f); total Akt, ERK, and GSK3β served as loading controls. g Cytokine-starved WT BMMs were treated with or without LY294002 (10 μM) or PD98059 (10 μM) for 16 h, and then a migration assay toward M-CSF (50 ng/ml) was performed. Right panel, percentage of migrated cells. h Cytokine-starved BMMs from WT and Pld2^−/− mice were treated with or without LY294002 (LY, 2.5 or 5 μM) for 16 h, and then a migration assay was performed. Right panel, percentage of migrated cells. Data are expressed as the mean ± SD. *p < 0.05. All scale bars represent 100 μm.