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PDGF D induces hBMSC osteoblastic differentiation. A and B , alkaline phosphatase (ALP) staining of hBMSCs cultured for 7 days ( A ) or 10 days ( B ) in growth or OB media containing CM from LNCaP (Neo) or LNCaP (D) without or with neutralizing AB CR002 or with rPDGF D proteins at indicated concentrations. C , RT–PCR analysis of ALP and HDAC2 (control). D , immunofluorescence staining of collagen I in hBMSCs cultured in growth or OB media with or without 1 nM rPDGF D for 3 days. E and F , Alizarin Red or von Kossa staining of hBMSCs cultured in the indicated media with or without 1 nM rPDGF D for 21 days ( E ). ALP, alkaline phosphatase; CM, conditioned media; hBMSC, human bone marrow mesenchymal stem cell; HDAC2, histone deacetylase 2; PDGF, platelet-derived growth factor; rPDGF, recombinant PDGF.

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase HUWE1 promotes PDGF D-mediated osteoblastic differentiation of mesenchymal stem cells by effecting polyubiquitination of β-PDGFR

doi: 10.1016/j.jbc.2022.101981

Figure Lengend Snippet: PDGF D induces hBMSC osteoblastic differentiation. A and B , alkaline phosphatase (ALP) staining of hBMSCs cultured for 7 days ( A ) or 10 days ( B ) in growth or OB media containing CM from LNCaP (Neo) or LNCaP (D) without or with neutralizing AB CR002 or with rPDGF D proteins at indicated concentrations. C , RT–PCR analysis of ALP and HDAC2 (control). D , immunofluorescence staining of collagen I in hBMSCs cultured in growth or OB media with or without 1 nM rPDGF D for 3 days. E and F , Alizarin Red or von Kossa staining of hBMSCs cultured in the indicated media with or without 1 nM rPDGF D for 21 days ( E ). ALP, alkaline phosphatase; CM, conditioned media; hBMSC, human bone marrow mesenchymal stem cell; HDAC2, histone deacetylase 2; PDGF, platelet-derived growth factor; rPDGF, recombinant PDGF.

Article Snippet: The induction of ALP by PDGF D-overexpressing LNCaP CM was abolished by the neutralizing antibody CR002 against PDGF D ( ) (a kind gift from Celldex Therapeutics), indicating the direct effect of PDGF D on osteoblastogenesis.

Techniques: Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Immunofluorescence, Histone Deacetylase Assay, Derivative Assay, Recombinant