Fig. 4: The MERTK-inhibitor R992 induces bone formation in healthy mice.

a Structure and chemical formula of R992: 3-(Butylamino)−5-((1r,4r)−4-hydroxycyclohexyl)−8-((4-methylpiperazin-1-yl)methyl)pyrimido[4,5-c]isoquinolin-6(5H)-one (C27H38N6O2). b Illustration of R992 docked in MERTK and the H-bonds R992 forms with three residues in the ATP pocket (MET-647, PRO-672, and ASP-741). c R992 on-target and off-target activity. d, e Immunoblots of MERTK phosphorylation and AKT signaling in human (d) and murine (e) cells. f μCT of the metaphyseal proximal region of tibias of 10-week-old healthy C57BL/6J mice treated after 2 weeks of treatment with vehicle or R992. g Quantification of trabecular bone volume (BV/TV) of the proximal tibia determined by μCT analysis (Vehicle n = 7 and R992 n = 7). h, i Representative pictures (h) and analysis (i) of bone formation rate by Calcein and Demeclocycline double labeling (Vehicle n = 7 and R992 n = 7). j, k Representative pictures of Alizarin red staining of calvarial cells cultures treated with different doses of R992 on day 21 (j). The number of mineralized nodules was quantified (k) (n = 3 biological replicates). l Analysis of osteoblast differentiation marker Alpl, Runx2, and Osx on day 7 in R992-treated calvarial cell cultures in comparison to control treated cultures (n = 3 biological replicates). Data were means ± SEM. Statistical significance was determined by a two-tailed unpaired t-test.