Fig. 5: CR4-WHD-tet inhibits RANKL-induced nuclear localization of p38, affecting the expression of differentiation marker genes.

a, b Effect of CR4-WHD-tet treatment on the nuclear localization of NFATc1 (a) and p38 (b). 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 (a, b, left panels). Nuclear localization was analyzed with immunocytochemistry using a specific antibody directed against NFATc1 (a, middle panel) or p38 (b, middle panel). Representative images of TRAP staining are shown (a). Scale bars, 50 µm. The percentage of nuclei with NFATc1 was measured (a, right panel; mean ± SEM, n [number of area with 5–31 nuclei] = 11–18, from 2 independent experiments). n.s., not significant (by two-sided Student’s t test). The percentage of nuclei with p38 was measured (b, right panel; mean ± SEM, n [number of area with 5–29 nuclei] = 14–19, from 3 independent experiments). ***P < 0.001 (by Tukey’s test). c Effect of CR4-WHD-tet treatment on the mRNA expression levels of genes involved in the late stage of differentiation. BMMs were cultured with RANKL in the presence or absence of 3 μM CR4-WHD-tet, as shown in the schematic diagram (upper panel). Relative amounts of each mRNA were determined with RT–qPCR using Gapdh as the reference gene with the amount of RANKL treatment only equal to 1 (n = 3–5, mean ± SEM, lower panels). *P < 0.05 (by Tukey’s test). n.s., not significant. If no expression levels were detected even after 40 cycles of amplification, the data were presented as zero.