Fig. 4: CR4-WHD-tet specifically inhibits p38 activation induced by RANKL in the late stage of differentiation.

a Effect of CR4-WHD-tet treatment on RANKL-induced osteoclastogenesis in each stage of differentiation. BMMs obtained from bone marrow cells were cultured with RANKL in the presence or absence of 3 μM CR4-WHD-tet, as shown in the schematic diagram (left panel). The early, middle, and late stages of differentiation correspond to 0-24 h, 24-48 h, and 48–72 h incubation, respectively. Data are presented as a percentage of the control value (without CR4-WHD-tet) (middle panel, n = 4, mean ± SEM). *P < 0.05 (by Tukey’s test). Representative images of TRAP staining are shown (right panel). Scale bar, 500 µm. b Effect of CR4-WHD-tet treatment on the mRNA and protein expression levels of NFATc1. BMMs were cultured with RANKL in the presence or absence of 3 μM CR4-WHD-tet, as shown in the schematic diagram (left upper panel). Relative amounts of NFATc1 mRNA were determined with reverse transcription–quantitative PCR (RT–qPCR) using Gapdh as the reference gene (n = 4–8, mean ± SEM, left lower panel). NFATc1 in the lysate was analyzed with western blotting, and the intensity of each band was quantified and presented as the amount of NFATc1 relative to β-actin (n = 3, mean ± SEM, right panels). n.s., not significant (by two-sided Student’s t test). c Effect of CR4-WHD-tet treatment on the activation of signaling molecules induced by RANKL in the late stage of differentiation. BMMs were cultured with RANKL, as shown in the schematic diagram. The cells were treated with or without 3 μM CR4-WHD-tet 30 min before the third treatment with RANKL. The cell lysate was analyzed with western blotting using specific antibodies, and the intensity of each band was quantified. Activation levels of p38 (upper left panel), JNK (lower left panel), and ERK (lower right panel) were evaluated as the amount of the phosphorylated form relative to the total protein. Activation levels of NF-κB were evaluated as the amount of the phosphorylated IκB (upper middle panel) and total IκB (upper right panel) relative to β-actin (n = 3–6; mean ± SEM). *P < 0.05 (by two-sided Student’s t test). d Effect of p38 inhibitor SB203580 treatment on RANKL-induced osteoclastogenesis in the late stage of differentiation. BMMs were cultured with RANKL, as shown in the schematic diagram. The cells were treated with or without 10 μM SB203580 after the third treatment with RANKL. Data are presented as a percentage of the control value without CR4-WHD-tet (n = 3, mean ± SEM). *P < 0.05 (by two-sided Student’s t test).