Fig. 3: Padi2 KO mice had CCD phenotype and showed the reduction of RUNX2 level.

A Whole-mount skeleton staining of Cont (n = 6) and Padi2 KO (n = 5) newborn littermates by Alizarin red and Alcian blue staining. Samples were cut into calvaria and clavicles. Scale bar: 2 mm. B Representative micro-CT images of the skull of postnatal day 7 (P7) Cont (n = 9) and Padi2 KO (n = 9) mice. Scale bar: 2 mm. C Representative micro-CT images of the skull of P7-old Cont (n = 6) and Padi2 ^Col1 (n = 5) mice. Scale bar: 2 mm. D The landmarks of the mouse clavicle for measuring the length of the clavicle (left). The clavicle length (a-a’) was measured and graphed in each sample in P7 Cont (n = 8) and Padi2 KO mice (n = 9) (right). E IHC for RUNX2 of the distal femur from 4-week-old Cont and Padi2 KO mice. The second and third rows show enlarged areas of metaphysis and cortical bone, respectively. Three independent experiments with three biological replicates for each group. Scale bar: 500 μm, 200 μm. F IHC for RUNX2 of distal femur from 4-week-old WT and Padi2^Col1 mice. The bottom shows cortical bone. Three independent experiments with three biological replicates for each group. Scale bar: 200 μm. G Primary calvarial OBs were cultured in an osteogenic medium for the indicated day, and RUNX2 and PADI2 levels were examined by western blot analysis. α-Tubulin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with α-Tubulin. H MC3T3-E1 cells were transfected with scrambled control siRNA (siCont), Padi2 siRNA #2 (siPadi2 #2), or Padi2 siRNA #3 (siPadi2 #3) and then cultivated in osteogenic media for additional 3 days. RUNX2 and PADI2 levels were examined by western blot analysis. α-Tubulin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with α-Tubulin. I RUNX2 and PADI2 levels in CRISPR–Cas9-mediated Padi2 KO cell clones (#3–4 and #5–6) and control cells were examined by western blot analysis. α-Tubulin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with α-Tubulin. J MC3T3-E1 cells were transfected with empty vector or Flag-PADI2 plasmids and then cultivated in osteogenic media for an additional 3 days. RUNX2 and Flag-PADI2 levels were examined by western blot analysis. GAPDH was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with GAPDH. Western blot data was collected from at least two or three independent experiments; the representative results are shown here.