Fig 2: 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 Padi2Col1 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.

Fig 3: Padi2 knockout mice exhibited reduced bone mass.A Physiognomy of Padi2 control (Cont), hetero (Het), and knockout (KO) litters at newborn and P7 (n = 4–6 in each group). B Representative micro-CT images of the distal femur at 4 months in the arterial view of a coronal section and cross-sectional views are shown for each genotype (scale bar:1 mm). C Histomorphometric analyses of 3D micro-CT data of control and Padi2 KO mice in both male (n = 9, Cont mice and n = 7, Padi2 KO mice) and female (n = 11, Cont mice and n = 12, Padi2 KO mice). BV/TV bone volume/tissue volume; Tb. Th trabecular thickness; Tb. N trabecular number; Tb. Sp trabecular separation. D, E Representative images of H&E stained distal femur and proximal tibiae from 4 months-aged control and Padi2 KO mice (scale bar: 500 μm, 200μm). F, G Representative images of immunohistochemistry (IHC) for PADI2 (F) and type 1 collagen (COL1) at 4 months-aged Cont and KO mice (G) (scale bar: 500 μm, 200 μm). H Representative images of TRAP-stained trabecular bone of distal femur from 4 months-aged Cont and KO mice. TRAP-positive purple spots indicate multinucleated osteoclasts (scale bar: 500 μm, 200 μm); We performed two or three independent experiments with three biological replicates for each group (D–H). I Osteoclast identification by TRAP staining. Bone marrow-derived macrophages (BMMs) isolated from Cont and KO mice were differentiated into osteoclasts in the presence of CSF1 (20 ng/ml) and RANKL (100 ng/ml) for 5 days (scale bar: 10 μm). The graph shows the number of TRAP-positive osteoclasts with more than three nuclei (n = 8 in each group). Three independent experiments with eight biological replicates for each group. Data are expressed as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N.S, not significant.

Fig 4: PADI2 protects RUNX2 from ubiquitin-proteasomal degradation pathway.A–C MC3T3-E1 cells were transfected with Strep-Runx2 with or without Flag-PADI2, and then cultivated in osteogenic medium for 3 days. On day 3, 4 μg/mL Actinomycin D was treated and incubated for 0, 3, and 6 h. The half-life of Runx2 mRNA and RUNX2 protein was determined by RT-qPCR (A) and western blot analysis (B), respectively. The intensities of Strep-RUNX2 protein levels were normalized against each GAPDH by ImageJ. The normalized values at 0 h were set as 1, and relative levels are shown (C). D, E MC3T3-E1 cells were transfected with Strep-Runx2 together with or without Flag-PADI2, and then cultivated in osteogenic medium for 3 days. 20 μg/mL cycloheximide was treated on the last day and incubated for 0, 3, and 6 h. The half-life of RUNX2 protein was determined by western blot analysis (D). The intensities of Strep-RUNX2 protein levels were normalized against each GAPDH by ImageJ and relative levels are shown (E). F Padi2 control and KO pOB cells were cultured in osteogenic media for 2 days, and 20 μM MG132 or DMSO as vehicle was treated for 6 h before harvesting cells, and western blot analysis followed. β-Actin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The red arrow indicates PADI2. G MC3T3-E1 cells were transfected with siCont or siPadi2 #2 and then cultivated in osteogenic media for an additional 2 days. 20 μM MG132 or DMSO as the vehicle was treated for 6 h before harvesting cells followed by western blot analysis. α-Tubulin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with α-Tubulin. The red arrow indicates PADI2. H hMSCs were transfected with siCont or siPADI2 and then cultivated in osteogenic media for an additional 2 days. 20 μM MG132 or DMSO as the vehicle was treated for 6 h before harvesting cells followed by western blot analysis. β-Actin was used as a loading control. RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The red arrow indicates PADI2. Western blot data were collected from at least two or three independent experiments; the representative results are shown here. I Strep-Runx2, Flag-PADI2, and HA-ubiquitin were transfected into 293 T cells. 3 days after transfection, cells were treated with 20 μM MG132 for 6 h, lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with anti-HA or anti-RUNX2 antibody. Ubiquination assay was performed in three independent experiments; the representative results are shown here.

Fig 5: Padi2 deficiency impaired osteoblast differentiation.A Representative view of 4-week-old female Cont and Padi2-KOCol1 mice. B Representative micro-CT images of the distal femur from 4-week-old female Cont and Padi2Col1 mice in midsagittal and coronal views. Scale bar: 500 μm. C Histomorphometric analysis of 3D micro-CT of the distal femur from data 4-week-old female Cont (n = 10) and Padi2Col1 (n = 6) mice. D IHC for PADI2 of distal femur from 4-week-old Cont and Padi2Col1 mice. Three independent experiments with three biological replicates for each group. Scale bar: 200 μm, 100 μm. E Representative images of H&E staining for distal femur from 4-week-old Cont and Padi2Col1 mice. Three independent experiments with three biological replicates for each group. Scale bar: 500 μm, 200 μm. F Relative mRNA expression of Padi2 and bone marker genes in Cont and KO primary calvaria osteoblasts (pOBs) cultured or not (Day0) in osteogenic medium for each indicated day as determined by RT-qPCR. Three independent experiments with three biological replicates for each group. G ALP and ARS staining were performed in Cont, Het, and KO primary calvaria OBs cultivated in an osteogenic medium for 5 days and 21 days, respectively. Representative images from three independent experiments with two biological replicates for each group. H TRAP staining of distal femurs from 4-week-old Cont and Padi2Col1 mice. The boxed region is shown in higher magnification (bottom). Three independent experiments with three biological replicates for each group. Scale bar: 500 μm, 200 μm. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N.S, not significant. ND not determined.