Fig. 4: Deletion of Dlk2 inhibits osteoclastogenesis in vitro by inhibiting Akt/ERK/p38 and interacting with Syap1.

A Representative images of TRAP-stained multinucleated osteoclasts. BMMs from Ctsk-Cre^-;Dlk2^fl/fl (WT) and Ctsk-Cre⁺;Dlk2^fl/fl (CKO) male mice were stimulated with 30 ng/ml M-CSF and 50 ng/ml RANKL for 5 days, fixed in 4% paraformaldehyde and stained with TRAP solution. The number and size of the TRAP⁺ multinucleated osteoclasts were quantified (Scale bar, 200 μm, n = 3 independent samples). B Representative images of osteoclastic bone resorption. BMM-derived osteoclasts were cultured on bone-mimicking plates for 9 days and removed by 2% sodium hypochlorite. The bone resorption area shown was quantified (Scale bar, 200 μm, n = 3 independent samples). C The proteins that interact with Dlk2 were identified by coimmunoprecipitation (co-IP) combined with liquid chromatography-mass spectrometry (LC-MS/MS) analysis. HEK-293T cells were transfected with 10 μg pLVX-CMV-3flag (control) and pLVX-CMV-Dlk2-3flag (Dlk2-ov) plasmids using polyethyleneimine solution for 48 h. Then, the cells were harvested with IP lysis buffer for 30 min and incubated with anti-Flag M2 magnetic beads at 4 °C for 8 h for coimmunoprecipitation. The immunoprecipitated proteins were identified by LC-MS/MS analysis. The related heatmap shows the top 15 proteins that coimmunoprecipitated with significantly greater abundance in Dlk2-ov group. D Interaction of Dlk2 and Syap1 in HEK-293T cells. Cells were transfected with pLVX-CMV-3flag (control) and pLVX-CMV-Dlk2-3flag (Dlk2-ov) plasmids. All the cellular extracts were incubated with anti-Flag beads (IP: Flag) and immunoblotted with an antibody against Syap1 (n = 3 independent samples). E Interaction of Syap1 and Dlk2 in HEK-293T cells. Cells were cotransfected with pLVX-CMV-3flag and pLVX-CMV-Dlk2-myc (control), and pLVX-CMV-Syap1-3flag and pLVX-CMV-Dlk2-myc plasmids. All the cellular extracts were incubated with anti-Flag beads (IP: Flag) and immunoblotted with an antibody against Myc (n = 3 independent samples). F Western blot analysis was performed to detect total and phosphorylated forms of Akt, ERK, JNK, and p38 in Ctsk-Cre^-;Dlk2^fl/fl (WT) and Ctsk-Cre⁺;Dlk2^fl/fl (CKO) osteoclasts induced by RANKL (n = 3 independent samples). G Representative images of immunofluorescence-stained femur sections from 8-week-old Ctsk-Cre^-;Dlk2^fl/fl (WT) and Ctsk-Cre⁺;Dlk2^fl/fl (CKO) male mice. The sections were permeabilized with 0.1% Triton X-100, blocked with 5% goat serum, incubated with primary antibodies against p-Akt^Ser473, p-ERK, p-p38 and Syap1 and then incubated with secondary antibodies. Nuclei were counterstained with DAPI (Scale bar, 20 μm, n = 3 independent samples). (Mean ± SD, Student’s tests, *p < 0.05, **p < 0.01).