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.