Fig. 5: Nf2-FAK interaction alteration impaired osteoblast differentiation.

a The Nf2 knockout primary osteoblasts (OB) at differentiation stage of D7 showed decreased activity of phospho-397FAK. b ALP staining exhibited that overexpression of Nf2 or FAK using lentivirus mediating system can sufficiently rescue the defective activities of Nf2 mutant osteoblasts. c The levels of phospho-397FAK, Runx2, Sp7 and collagen type I were sufficiently rescued after the lentivirus mediated Nf2 overexpression in Nf2 mutant osteoblasts. d co-IP assay showed that FLAG-FAK and HA-NF2 forms an interaction in 293 T. e co-IP assay confirms that Nf2 and FAK interacted in primary osteoblasts. f, g NanoBit protein:protein interaction system (N2015, Promega) using paired Nf2 and FAK plasmids based on structural conformation (f) showed that C-terminal domain of Nf2 protein can physically interact with the N-terminal domain of FAK in osteoblast cell line MC3T3-E1(3T3)(g). g The bioluminescence ratios between NF2-FAK interaction and negative control (n = 3) were analyzed using Nano-Glo® Live-cell Reagent (N1661, Promega) (Y: bioluminescence value; X: paired plasmids, such as N represents NF2; F represents FAK; C represents C terminal; N represents N terminal; 1.1 represents LgBiT, 2.1 represents SmBiT). h The immunosignals of phospho-FAK (397) were largely diminished in Runx2⁺ osteoprogenitor in Nf2 mutants at E12.5 coronal section of the skull (arrow). i The immunosignals of phospho-FAK (397) were significantly reduced in Nf2 mutants at E18.5 sagittal section of the skull. Scale bar: 20 μm in (b) 50 μm in (h, i). Data were expressed as means ± SD and each dot represents an individual biological replicate. P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments. *p < 0.05. **p < 0.01, ***p < 0.001.