Wan et al. 10.1073/pnas.0604849103. |
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Materials and Methods
Supporting Figure 11
Fig. 7. Gene analysis confirms ADAS differentiation capacity. QRT-PCR analysis revealed no up-regulation in markers for osteogenic differentiation after 21 days in ODM (n = 3 for each differentiated ADAS sample) (A), whereas ADAS cultured in adipogenic media for 5 days were observed to significantly up-regulate both PPARg and adipsin (*, P < 0.05; n = 3 for each differentiated ADAS sample) (B).
Fig. 8. RA and BMP signaling pathways are present in ADAS. RT-PCR analysis reveals the presence of transcripts for all six retinoic acid receptor isoforms in ADAS (A). Note that, although faint, a band for RXRb is present. All three BMP receptor isoforms were also found to be expressed (B). In addition, transcripts were noted for both Smad1 and Smad5, confirming the presence of BMP signaling intermediates in ADAS (C).
Fig. 9. RA and/or BMP-2 impair ADAS proliferation. Proliferation rates were significantly reduced when ADAS were cultured in the presence of RA or RA with BMP-2 (*, P < 0.05; n = 3 counts for each group). Note the significant difference in cell number increase at day 7.
Fig. 10. RA-induced osteogenesis is limited to cells with osteogenic potential. The effect of RA on osteogenesis was not observed in 3T3-L1 preadipocytes or fibroblasts. Minimal von Kossa staining was noted when 3T3-L1 or primary fibroblasts were cultured in ODM with both RA and BMP-2. Quantification of staining by using Scion Image Analysis is noted in the upper right corner of each well.
Fig. 11. BMPR-IB siRNA construct sequences. Shown is a schematic of the BMPR-IB transcript (NM_007560) along with sequences of target siRNA constructs designed.
Proliferation Assay.
After plating, ADAS were cultured by using standard growth media supplemented with 2.5 mM RA and/or 50 ng/ml rhBMP-2. Cells were then trypsinized every other day up to 7 days total, and the cell number was determined by counting with a hemocytometer. Experiments were all performed in triplicate.RT-PCR and QRT-PCR.
Primers were designed with the assistance of PrimerBank for each of the following target mRNA transcripts: Runx2, alk phos, Opn, Ocn, PPARg, adipsin, BMPR-IA, BMPR-IB, and BMPR-II (1). Sequences of each primer and probe were confirmed to be specific for the target gene by using NIH BLAST and gel electrophoresis. RT-PCR was performed by using PCR Master Mix (Promega, Madison, WI) to evaluate for expression of BMP and RA receptors. QRT-PCR analysis was performed by using a two-step, multiplexed TaqMan 5'®3' exonuclease assay (2). Each cDNA sample was evaluated for transcript levels (including the normalization control glyceraldehyde-3-phosphate dehydrogenase) in triplicate by using a SYBR Green assay and quantified with the ABI Prism 7900HT Sequence Detection System from Applied Biosystems (Foster City, CA).Retroviral Generation.
Short-hairpin constructs with an intervening loop sequence of -TCTCTTGAA- were synthesized and cloned into a pSuper.retro.puro vector plasmid (Oligoengine, Seattle, WA). Ecotropic phoenix packaging cells (gift from Garry Nolan, Stanford University, Stanford, CA) were transfected by using FuGENE6 (3 ml per 1 mg of plasmid) (Roche, Basel, Switzerland). Medium containing infection-competent retroviruses was collected 48 h after initial transfection (3). Hexadimethrine bromide (5 mg/ml) was used to augment infection efficiency of ADAS.Immunofluorescent Staining.
Cells were fixed with 4% paraformaldehyde and washed with 0.1% Triton X-100 and cold acetone. Cells were incubated overnight at 4°C with a polyclonal goat anti-BMPR-IB antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a fluorescein-conjugated donkey anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. Hoechst counterstaining was performed on all cells, and a Zeiss Axioplan microscope was used for imaging.Western Blot Analysis.
For whole-cell protein Western blot analysis, cells were lysed with RIPA buffer and proteins were isolated by centrifugation at 14,000 ´ g for 30 min. For membrane fraction protein isolation, cells were homogenized with a Dounce homogenizer (Kontes, Vineland, NJ) in 10 mM Tris•HCl (pH 7.4). The nuclear fraction was then pelleted by centrifugation at 500 ´ g for 10 min. Membrane-associated proteins were then isolated by centrifugation of the resultant supernatant at 16,000 ´ g for 40 min. Smad Western blotting was performed by using 50 mg of protein, and BMPR-IB Western blotting was performed by using 80 mg of protein loaded onto a 10% SDS/polyacrylamide gel (Bio-Rad, Hercules, CA). Proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) and probed overnight at 4°C with primary antibodies [monoclonal mouse anti-Smad1 (Santa Cruz Biotechnology), polyclonal rabbit anti-Smad5 (Cell Signaling Technology, Beverly, MA), or polyclonal goat anti-BMPR-IB (Santa Cruz Biotechnology)]. Incubation with an appropriate HRP-linked secondary antibody and enhanced chemiluminescence were used to detect for the presence of protein (Amersham Pharmacia, Sunnyvale, CA). Coomassie blue PhastGel (GE Healthcare, Piscataway, NJ) staining was used to confirm equivalent loading of protein in each lane. A 30% methanol/10% acetic acid destaining solution was used to remove background stain, allowing for visualization of protein bands.1. Wang, X. & Seed, B. (2003) Nucleic Acids Res. 31, e154.
2. Mathy, J. A., Lenton, K., Nacamuli, R. P., Fong, K. D., Song, H. M., Fang, T. D., Yang, G. P. & Longaker, M. T. (2003) Plast. Reconstr. Surg. 112, 528-539.
3. Sen, G., Wehrman, T. S., Myers, J. W. & Blau, H. M. (2004) Nat. Genet. 36, 183-189.