Fig. 5: RANKL-induced osteoclastogenesis is regulated by p53-AKT-FOXO3.

a IF staining of p53 (green) and pFOXO3 (red) was performed on the subchondral bone of joints from mice fed with an HFD. n = 6 mice per group. Scale bar, 100 µm. b, c Representative images of TRAP staining (b) and quantification of mature TRAP⁺ osteoclasts (c) were performed after culturing with PFT β (10 μM), MK-2206 (10 μM), or infection with lentivirus expressing FOXO3, followed by treatment with RANKL for 6 days (n = 4-6). Scale bar, 100 µm. d The mRNA expression levels of Acp5, Atp6v1h, Ctsk, Dcstamp, Fos, and Nfatc1 in mouse osteoclasts. During the differentiation of BMDMs into osteoclasts, a combination of the p53 inhibitor PFT β (10 μM) and treatment with RANKL for 6 days was applied. n (Control) = 3, n (PFT β) = 3. e The mRNA expression levels of Acp5, Atp6v1h, Ctsk, Dcstamp, Fos, and Nfatc1 in mouse osteoclasts. During the differentiation of BMDMs into osteoclasts, a combination of the AKT inhibitor MK-2206 and treatment with RANKL (100 ng/ml) for 6 days was applied. n (Control) = 3, n (MK-2206) = 3. f The mRNA expression levels of Acp5, Atp6v1h, Ctsk, Dcstamp, Fos, and Nfatc1 in mouse osteoclasts. n (Lv-NC) = 3, n (Lv-FoxO3a) = 3. Mouse BMDMs were transfected with lentivirus expressing FOXO3 or an empty vector, followed by treatment with RANKL (100 ng/ml) for 6 days. Data are represented as mean ± s.d. Two-tailed Student’s t tests was performed. All exact p-values have been indicated in the figures. For RT-qPCR in (d–f), GAPDH was utilized as the control. Source data are provided as a Source Data file.