Journal: Stem cell research & therapy

Article Title: Centrifugal gravity-induced BMP4 induces chondrogenic differentiation of adipose-derived stem cells via SOX9 upregulation.

doi: 10.1186/s13287-016-0445-6

Figure Lengend Snippet: Fig. 3 CG induced SOX9 upregulation via increased expression BMP4. a Expression of BMP2, BMP4, and TGF-β1 mRNAs in CG-stimulated ASCs. ASCs were loaded with different degrees of CG (0, 300, 600, 1200, and 2400 g) for 15 min. b The duration of BMP4 mRNA expression in CG-stimulated ASCs. c Expression of BMP4 protein in CG-stimulated ASCs. ASCs were centrifuged at 2400 g for 30 min and then harvested at the indicated time points. Expression of BMP4 mRNA and protein was examined by RT-PCR and western blotting, respectively. d BMP4-dependent SOX9 expression in CG- stimulated ASCs. ASCs were stimulated with CG (2400 g for 30 min) and treated with recombinant BMP4 (10 nM) or Dorsomorphin (25 nM). SOX9 protein expression was examined using western blotting. The intensities of the bands in western blots were determined using ImageJ 1.40. BMP bone morphogenetic protein, CG centrifugal gravity, SOX SRY (sex-determining region Y)-box, TGF transforming growth factor

Article Snippet: Protein lysates (30 μg) and concentrated supernatants were resolved by 10–12% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then probed with primary antibodies against SOX9 (EMD Millipore, Billerica, MA, USA), BMP4 (Santa Cruz Biotechnology, Inc., Dallas, Texas USA), and GAPDH (Abcam).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Recombinant

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 1. Characteristics of NIH/3T3 and C2C12 cells. (A) Immunostaining for desmin expression by NIH/3T3 cells was negative. (B and C) We used immunostaining to monitor vimentin expression by NIH/3T3 cells stimu- lated with BMP4 (500 ng/ml). NIH/3T3 cells expressed vimentin on day 0 but exhibited decreased expression of vimentin over time when stimulated with BMP4. (D and E) Flow cytometry results showed that large percent- ages of NIH/3T3 and C2C12 cells co- expressed CD34 and Sca-1.

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques: Immunostaining, Expressing, Flow Cytometry

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 2. Chondrogenic differentiation of NIH/3T3 and C2C12 cells in pellet cultures treated with BMP4 (500 ng/ml) for 14 and 21 days. (A) NIH/3T3 cells were Alcian blue positive on days 14 and 21. (B) NIH/3T3 pel- let cultures exhibited type II collagen expres- sion on day 14, with a more intense brown signal on day 21. (C) C2C12 pellet cultures were Alcian blue negative and (D) did not express type II collagen.

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques:

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 3. ALP staining reveals that NIH/3T3 cells treated with BMP4 underwent osteogenic differentiation in a dose- and time- dependent manner. (A) Percentages of ALP+ cells in C2C12 and NIH/3T3 cell cultures incubated with various doses of BMP4 for 5 days. (B) Comparison of the percentages of ALP+ cells in NIH/ 3T3 and C2C12 cell lines cultured in the presence of BMP4 (500 ng/ml) for various periods of time. (C) NIH/3T3 cells became ALP+ after treatment with BMP4 (500 ng/ml) for 9 days. (D) We used RT-PCR to visualize the time sequence of osteocalcin gene expression in NIH/3T3 cells treated with BMP4 (500 ng/ml). A time-dependent increase in osteocalcin expression was observed in NIH/3T3 cells after BMP4 stimulation.

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques: Staining, Incubation, Comparison, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Gene Expression, Expressing

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 4. NIH/3T3 and C2C12 cells display differential chondrogenic and osteogenic potentials in vivo. (A) NIH/3T3-L-B cells and (B) C2C12-L-B cells were transduced efficiently by retro-LacZ viruses. (C) Results of the BMP4 bioassay show the amount of BMP4 produced by the C2C12-L-B, C2C12-L-B-D, and NIH/3T3-L-B cells. Staining for LacZ and eosin in both the (D) NIH/3T3-L-B and the (E) C2C12-L-B groups reveals LacZ+ cells in the newly formed bone (arrows). Double staining for LacZ and osteocalcin shows LacZ+

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques: In Vivo, Bioassay, Produced, Staining, Double Staining

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 5. Histological analysis of tissues harvested from SCID mice 28 days after intramuscular implantation of C2C12-L-B or NIH/3T3-L-B cells. Von Kossa and eosin staining shows (A) a small amount of mineralized bone formation in the NIH/3T3-L-B cell group and (B) a large amount of mineralization in the C2C12- L-B cell group. (C) Alcian blue and eosin staining reveals exten- sive cartilage formation in the NIH/3T3-L-B cell group; those results contrast with the results observed in the (D) C2C12-L-B cell group. (E) Western blot for the Runx2 protein shows that Runx2 was present at a very low level in the NIH/3T3 and NIH/ 3T3-L-B cells and at high levels in the C2C12 and C2C12-L-B cells 1 day after transduction with the retrovirus containing the BMP4 gene.

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques: Staining, Western Blot, Transduction

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 6. Transduction of C2C12 cells with a retrovirus encoding DNRunx2 impaired endochondral bone formation. (A) Western blot analysis confirmed the expression of DNRunx2 in the C2C12 cells transduced with retro-DNRunx2. NIH/3T3 cells transduced to express BMP4 and cultured for 14 days began to express increased levels of Runx2 protein. (B) X-ray analysis revealed less bone and delayed bone formation at different time-points in the mice that received C2C12-L-B-D cells than in the mice that received C2C12-L-B cells. However, the former cells still displayed more rapid and more robust bone formation than did NIH/3T3-L-B cells. (C) Twenty days after implantation, the relative area of the newly formed bone (data based on X-ray analysis) was significantly smaller (p < 0.01) in the group that received C2C12-L-B cells transduced with the DNRunx2 retrovirus (C2C12-L-B-D) than in the group that received C2C12-L-B cells. However, the C2C12-L-B-D cell group still displayed significantly more bone formation than the NIH/3T3-L-B cell group (p < 0.01). (D) Stainings of in vivo sections showed that the C2C12-L-B cell group exhibited human BMP4 expression at levels similar to those of the C2C12-L-B-D cell group at the various time-points and that both C2C12 cell groups showed stronger expression of human BMP4 than observed in the NIH/3T3-L-B cell group.

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques: Transduction, Western Blot, Expressing, Cell Culture, In Vivo

Journal: Journal of Bone and Mineral Research

Article Title: Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

doi: 10.1359/jbmr.050513

Figure Lengend Snippet: FIG. 9. Results obtained from experiments based on primary fibroblasts and myoblasts. (A) Differential expression of both desmin and vimentin by primary fibroblasts and myoblasts. (B) Results of ALP staining reveal more ALP+ cells in the primary myoblast group than in the primary fibroblast group after treatment of both cell groups with various doses of BMP4 for 2 days. (C) Results of the cell proliferation assay show that C2C12 cells proliferate at significantly higher rates than NIH/3T3 cells after stimulation of both groups with various doses of BMP4 (p < 0.01). There was no significant difference between the cell proliferation rates of primary fibroblasts and primary myoblasts after treatment with various doses of BMP4 (p 0.95). Overall, the proliferation potential of the primary cells was lower than that of the cell lines. (D) Results of the BMP4 bioassay show different amounts of BMP4 produced by the two groups. (E) Histological results obtained at different time-points after implantation show that PM-L-B cells induced more rapid endochondral bone formation that yielded more bone than did PF-L-B cells. (F and G) Quantitative measurement of the areas of bone and cartilage (in pixels) show that PM-L-B cells produced more bone than PF-L-B cells; there was no difference in the amount of cartilage formed by the two groups at different time-points after cell implantation. PF, primary fibroblasts; PM, primary myoblasts; PF-L-B, primary fibroblasts co-transduced with retro-LacZ and retro-BMP4 viruses; PM-L-B, primary myoblasts co-transduced with retro-LacZ and retro-BMP4 viruses.

Article Snippet: Human BMP4 antibody (1:100 dilution, AF757; R&D Systems) was used for the immunostaining.

Techniques: Quantitative Proteomics, Staining, Proliferation Assay, Bioassay, Produced, Transduction

Journal: Annals of surgery

Article Title: Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett’s Esophagus to Invasive Esophageal Adenocarcinoma

doi: 10.1097/SLA.0000000000003455

Figure Lengend Snippet: Analysis of mRNA expression of SKGT4 cells following addition of multiple concentrations of itraconazole. Analysis of mRNA expression of SKGT4 cells following addition of multiple concentrations of itraconazole (0, 1, 5, 10μM). RNA was extracted at the indicated hour after itraconazole addition and analyzed by qRT-PCR for mRNAs encoding bone morphogenic protein 4 (BMP4). The results were normalized to 0μM of itraconazole at each time point. *P < 0.05 compared to 0μM, **P < 0.005 compared to 0μM, Student t test, mean ± SD, n = 6 independent cell culture for each condition at 24 hours and n = 4 independent cell culture for each condition at 48 and 72hours.

Article Snippet: Slides were incubated with a rabbit primary antibody to either SHH (Novus Biologicals), IHH (Abcam, Cambridge, MA), BMP4 (Atlas Antibodies, Bromma, Sweden), or Sry-Box 9 (SOX9; Millipore-Sigma, St. Louis, MO) for 45 minutes followed by an incubation with PowerVision Poly-HRP anti-Rabbit IgG (Leica Biosystems, Chicago, IL) for 30 minutes.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture