Fig 6. Stable overexpression of ΔNp63α and ΔNp63γ increased the mRNA expression of osteocalcin and osterix, and decreased VDR.

hMSC were transfected with pcDNA3.0-p63 vectors (7 days), followed by selection with G418 (3 weeks). The cells were then expanded and cloned over 2 months. Overexpression of TAp63β, TAp63γ and ΔNp63β caused a cell proliferation arrest; hence they were not available for long-term expansion and analysis. A) RT-qPCR analysis of p63 isoform (left panels, TA- and ΔNp63) and splice variant mRNA (right panels, p63α / β and p63γ) expression. RT-qPCR analysis demonstrated the relative level of overexpression of each of the p63 variants compared to empty vector (pcDNA3.0). B) RT-qPCR analysis of mRNA expression of osteoblastic differentiation markers: VDR (vitamin D₃ receptor (binds 1α,25-dihydroxyvitamin D₃)), Osteocalcin, and Osterix. Also of note, the overexpression of TAp63α, ΔNp63α, and ΔNp63γ did not alter the mRNA expression of runx2, osteopontin or the late stage osteoblastic gene, BSP. N = 3 independent experiments in triplicate. (*) p ≤ 0.05 compared to control (hMSC with empty vector, pcDNA3.0). hMSC used were from a 22 year old male.