The human TWIST1 cDNA (Open Biosystem, Rockford, IL, USA) was subcloned into the EcoRI and HincII sites of the pWPI lentiviral vector (Addgene, Cambridge, MA, USA). The empty pWPI vector, encoding enhanced green fluorescent protein (EGFP), was used as the control. Two TWIST1 silencing vectors, expressing different short hairpin RNA (shRNAmir) sequences targeting different segments of human TWIST1 mRNA, were obtained from Open Biosystem. The silencing effects of each shRNA on the target gene were determined by Western blot analysis of target protein expression following infection of DPSC cells with shRNA-expressing lentiviruses (see below). The shRNA sequence yielding the highest suppression level against the target gene was used for following studies. The empty pGIPZ silencing vector, expressing Turbo green fluorescent protein (tGFP), from Open Biosystem, was used as control.
To generate the pDspp-luc construct, we released a 5.7-kb promoter fragment of Dspp
gene from the pBS II SK+ vector (provided by Dr. Ashok Kulkarni, NIH/NIDCR) by XbaI and NruI digestion and subcloned it into the NheI and HindIII sites of the pGL3-basic vector (Promega, Madison, WI, USA). The pDmp1-luc construct, containing a 9.6-kb promoter region, exon 1 and intron 1 of the Dmp1
gene, was prepared as described earlier (Lu et al., 2005
). The human RUNX2 cDNA, encoding FLAG-RUNX2, was obtained from Dr. Brendan Lee.
Culture of DPSCs
DPSCs isolated from adult human third molars were kindly provided by Dr. Songtao Shi at the University of Southern California. DPSCs were maintained in growth medium, containing alpha modification of Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal calf serum (Hyclone, Logan, UT, USA), 100 µg/mL L-ascorbic acid 2-phosphate (Sigma, St. Louis, MO, USA), 2 mmol/L glutamine (Invitrogen), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Invitrogen). For differentiation assays, DPSCs were grown to confluence in growth medium, then in differentiation medium containing normal growth medium supplemented with 100 µg/mL L-ascorbic acid 2-phosphate, 2 mM β-glycerophosphate, and 10 nM dexamethasone for 2 or 4 wks.
293FT cells (Invitrogen) were maintained in DMEM supplemented with 10% fetal bovine serum (Hyclone), 2 mmol/L glutamine (Invitrogen), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Invitrogen). To prepare lentivirus for DPSC cell infection, we co-transfected 293FT cells with lentiviral vector, packaging vector psPAX2, and envelope vector pMD2.G, using Fugene6 (Roche, Indianapolis, IN, USA), as per the manufacturer’s instructions. The supernatant was collected 48 hrs after transfection and filtered with 0.45-µm filters for removal of cell debris.
Lentiviral Transduction and Cell-sorting
For lentiviral transduction, sub-confluent DPSCs were incubated with lentiviral supernatant in the presence of 10 µg/mL of polybrene (Sigma) for 48 hrs, with lentiviral supernatant changed once. DPSCs stably expressing green fluorescent protein (GFP) were selected by fluorescent-activated cell-sorting (FACS), with the use of a Vantage SE Diva cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Sorted DPSCs were expanded for two passages for further studies. Cells overexpressing or silencing TWIST1 were confirmed by Western blot analysis.
Western Blot Analysis and von Kossa Staining
Blots were first immunolabeled with the following primary antibodies, anti-TWIST1 monoclonal antibody (Abcam, 1:50), anti-alkaline phosphatase monoclonal antibody (Abcam, Cambridge, MA, USA; 1:100), anti-osteocalcin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), rabbit anti-dentin matrix protein 1 polyclonal antibody (gift from Larry W. Fisher, 1:500), anti-osteopontin monoclonal antibody (Santa Cruz Biotechnology, 1:1000), rabbit anti-dentin sialoprotein polyclonal antibody (gift from Larry W. Fisher, 1:250), or anti-β-actin monoclonal antibody (Sigma, 1:20,000). They were then incubated with horseradish-peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (Santa Cruz Biotechnology). The blot was finally detected with ECL reagents (GE Healthcare Bio-Sciences, Piscataway, NJ, USA).
For von Kossa staining, DPSC cells were fixed with 4% paraformaldehyde-0.1M phosphate buffer, and stained with 10% silver nitrate for von Kossa staining.
We used promoter-luciferase assays to determine the effects of RUNX2 and TWIST1 interactions on Dmp1 and Dspp promoter activities in 293FT cells. The cells were transiently transfected with pDmp1-Luc or pDspp reporter construct and empty vector or vectors expressing FLAG-RUNX2 or TWIST1 or both, with Fugene 6 as described above. pRL-TK was co-transfected as an internal control for normalizing transfection efficiency. Firefly and Renilla luciferase activities were analyzed 48 hrs after transfection with the use of a dual luciferase assay system (Promega) following the manufacturer’s instructions. Each assay was performed in triplicate and repeated at least three times.