Fig. 3

EP4 deletion in osteoclasts reduces pain in OA by suppressing Netrin-1 secretion and CGRP-positive sensory neuron recruitment to subchondral bone. Von Frey assay and thermal hyperalgesia test conducted at the right hind paw of the EP4^fl/fl or littermate EP4^LysM mice 2 weeks (a) or 8 weeks (b) after ACLT surgery. Error bars are the mean ± s.d. Two-way ANOVA followed by Tukey’s t tests. n = 6 for each group. c qRT-PCR analysis of the mRNA levels of Ngf and Netrin-1 in osteoclasts generated from BMMs stimulated with 10 ng·mL⁻¹ M-CSF and 50 ng·mL⁻¹ RANKL and incubated with 100 nmol·L⁻¹ PGE2 for 5 days. The experiments were performed with three biological replicates. Error bars are the mean ± s.d. **P < 0.01 and ns, not significant by one-way ANOVA followed by Tukey’s t tests. d Representative images of Netrin-1 protein by western blotting of osteoclasts generated using BMMs from the EP4^fl/fl or littermate EP4^LysM mice stimulated with 10 ng·mL⁻¹ M-CSF and 50 ng·mL⁻¹ RANKL and incubated with 100 nmol·L⁻¹ PGE2 for 5 days. The experiments were performed with three biological replicates. Representative images of IHF staining of Netrin-1 (e, red) and CGRP-positive sensory nerve fibers (f, red) in the subchondral bone of the EP4^fl/fl or littermate EP4^LysM mice 2 weeks after ACLT surgery (left) and corresponding quantitative analysis (right). The white arrows indicate Netrin-1 or CGRP IHF signals. Error bars are the mean ± s.d. Two-way ANOVA followed by Tukey’s t tests. n = 3 for each group. Scale bars, 20 μm