Fig. 4: The SGI-1776 Pim1 inhibitor downregulates osteoclast resorption by blocking cytoskeleton reorganization.

BMMs were incubated with SGI-1776 (0.1, 0.3 or 1 nM) for 4 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml). a The cells were cultured as described in wells and then stained with TRAP. Left: representative images of the osteoclasts. Scale bars, 200 μm. Right: plot showing the average number of TRAP-positive multinucleated cells with ≥200-μm diameters. b The cells were cultured as described on dentin and then stained with hematoxylin. Left: representative images of the osteoclasts. Scale bars, 200 μm. Right: plot showing the average resorption-pit area. c The cells were cultured as described on dentin slices and stained with Alexa Fluor 488-phalloidin. Left: representative images of the osteoclasts. Scale bars, 200 μm. Right: plot showing the average frequency of osteoclasts with a normal actin ring. d The cells were cultured as described on glass slides, incubated with antibodies specific for tubulin and acetylated tubulin followed by Alexa Fluor 594 (bright red)- and Alexa Fluor 488 (green)-labeled secondary antibodies, respectively. The cells were also stained with Alexa Fluor 647-phalloidin (green) to indicate their F-actin. Left: representative images. Scale bars, 20 μm. Right: mean intensity of acetylated tubulin per osteoclast (n = 10) (right). All data are representative of three independent experiments. The data are presented as mean ± s.d. Statistical analysis was conducted with one-way ANOVA with Tukey’s multiple comparison test. ns, not significant. ***P < 0.001, ****P < 0.0001.