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Inhibition of rhBMP‐2‐induced ALP activity by intracellular delivery of SMURF1 in murine calvarial preosteoblast cells
Author(s) -
Hsu ChiaWei,
Liu Shiguang,
Hsu Eric,
Hollinger Jeffrey O.
Publication year - 2014
Publication title -
journal of biomedical materials research part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.849
H-Index - 150
eISSN - 1552-4965
pISSN - 1549-3296
DOI - 10.1002/jbm.a.35046
Subject(s) - materials science , intracellular , biophysics , microbiology and biotechnology , cancer research , biology
Intracellular protein delivery is a novel tool for functional analysis of protein inside a cell. Several protein delivery reagents with diverse mechanisms have been developed and are commercially available. In this study, we focused on the inhibitory effect of intracellular delivery of SMAD ubiquitination regulation factor 1 (SMURF1) on the recombinant human bone morphogenetic protein‐2 (rhBMP‐2) signaling pathway. First, three commercially available reagents for intracellular delivery (BioPORTER ® , PULSin ® , and Xfect TM ) were tested in a murine preosteoblast cell line, MC3T3‐E1.4. The biocompatibility of these reagents was examined by 3‐(4,5‐dimethylthiazol‐2‐yl)−5‐(3‐carboxymethoxyphenyl)−2‐(4‐sulfophenyl)−2H‐tetrazolium assay and the cellular uptake and delivery efficiency were determined with FITC‐antibody and β‐galactosidase (β‐gal), respectively. BioPORTER ® provided the best results and was, therefore, chosen for the second aspect of our study: intracellular SMURF1 delivery. SMURF1/BioPORTER ® complexes were applied to cells prior to rhBMP‐2 application. The outcome data suggest intracellular SMURF1 delivery in MC3T3‐E1.4 cells significantly inhibited alkaline phosphatase upregulation. This outcome may be useful to off‐targets effects of rhBMP‐2. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4037–4043, 2014.