Fig. 3: Pim1 promotes osteoclast cytoskeleton reorganization by inducing microtubule acetylation.

a–c, WT and Pim1^−/− BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4–6 days on dentin slices or glass slides: the cells on the dentin slices were stained with Alexa Fluor 488-phalloidin (left: representative images of the osteoclasts (scale bars, 200 μm); right: plot of the frequency of osteoclasts that bore an actin ring) (a); the cells on the glass slides were stained with Alexa Fluor 594-phalloidin (left: representative images of the osteoclasts (scale bars, 20 μm); right: plot of the frequency of osteoclasts that bore a podosome belt) (b); the cells on the glass slides were incubated with antibodies specific for tubulin or acetylated tubulin followed by Alexa Fluor 594 (bright red)- and Alexa Fluor 488 (green)-labeled secondary antibodies, respectively, and F-actin was also stained with Alexa Fluor 647-phalloidin (purple) (left: representative images (scale bars, 20 μm); right: mean intensity of acetylated tubulin per osteoclast (n = 10)) (c). d WT and Pim1^−/− BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) in wells for the indicated durations, and the total cell lysates were collected and immunoblotted with the indicated antibodies. Band intensity was determined with ImageJ. Left: representative immunoblot. Right: average amount of acetylated tubulin relative to total tubulin. All data are representative of three independent experiments. The data are presented as mean ± s.d. Statistical analysis was conducted with two-tailed unpaired Student’s t-test and two-way ANOVA with Tukey’s multiple comparison test. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.