Journal: Scientific Reports

Article Title: Cell proliferation by silk gut incorporating FGF-2 protein microcrystals

doi: 10.1038/srep11051

Figure Lengend Snippet: ( A ) Detection of the recombinant protein in posterior silk glands. Protein samples (7 μl) from w1-pnd strain (lane 1), BmFibH-polyhedrin line (lane 2), BmFibH-H1/FGF-2 line (lane 4), and BmFibH-polyhedrin/H1/FGF-2 line (lane 5) posterior silk glands on the 6th day of the 5th instar were electrophoresed in a 12.5% gel with a molecular marker (lane M), rhFGF-2 (lane 3), and polyhedron-encapsulated H1/FGF-2 prepared from cultured Sf21 cells (lane 6); FGF-2 (upper panel) and polyhedrin (lower panel) were detected by immunoblotting using anti-polyhedrin and anti-FGF-2 antibodies as described in the Methods section. Degraded H1/FGF-2 bands (approximately 17 kDa) were observed in lane 6 (upper panel). Arrows indicate polyhedrin, H1/FGF-2, and rhFGF-2. Original gel images of the data are presented in . ( B ) Polyhedra formed in the posterior silk glands of BmFibH-polyhedrin and BmFibH-polyhedrin/H1/FGF-2 lines were fixed on a glass-based dish and the H1/FGF-2 encapsulated within the polyhedra was detected by immunofluorescence as described in the Methods section. Bar, 50 μm.

Article Snippet: Commercial recombinant human FGF-2 (rhFGF-2; R&D Biochemicals, Inc.) and H1/FGF-2-encapsulating polyhedra prepared from Sf21 cell culture were treated as above.

Techniques: Recombinant, Marker, Cell Culture, Western Blot, Immunofluorescence

Journal: Scientific Reports

Article Title: Cell proliferation by silk gut incorporating FGF-2 protein microcrystals

doi: 10.1038/srep11051

Figure Lengend Snippet: ( A ) Detection of phosphorylated p44/p42 mitogen-activated protein kinase (MAPK) in NIH3T3 cells. Silk gut powders (1 mg/well) from the w1-pnd strain as a non-transgenic negative control (lane 4), from the BmFibH-polyhedrin line as a transgenic negative control (lane 5), and from lines BmFibH-H1/FGF-2 (lane 6) and BmFibH-polyhedrin/H1/FGF-2 (lane 7) were placed on the inside filters of cell culture inserts, which were then introduced into the medium of serum-starved NIH3T3 cells. Additionally, cells were not treated (lane 3; negative control) or similarly treated with 10 ng/well of rhFGF-2 (positive control; lane 1) or 1 × 10 5 cube/well of H1/FGF-2-encapsulating polyhedra from Sf21 (positive control; lane 2). After 4 h of cultivation, cell lysate samples (5 μg proteins) were analysed by immunoblotting using either p44/p42 MAPK antibody or phosphorylated p44/p42 MAPK antibody. Original gel images of the data are presented in . ( B ) Relative cell numbers were measured by WST-8 assay. After starvation, cells were cultured in assay medium containing either rhFGF-2 or cell culture inserts containing the indicated amounts of silk gut powders from w1-pnd strain, BmFibH-polyhedrin line, BmFibH-polyhedrin/H1/FGF-2 line or BmFibH-H1/FGF-2 line. After incubation for 48 h at 37 °C under 5% CO 2 , cell numbers were measured by WST-8 assay. Absorbance at 450 nm was determined by using a micro-plate reader and mean values of triplicate experiments are presented with standard deviations.

Article Snippet: Commercial recombinant human FGF-2 (rhFGF-2; R&D Biochemicals, Inc.) and H1/FGF-2-encapsulating polyhedra prepared from Sf21 cell culture were treated as above.

Techniques: Transgenic Assay, Negative Control, Cell Culture, Positive Control, Western Blot, Incubation

Journal: Scientific Reports

Article Title: Cell proliferation by silk gut incorporating FGF-2 protein microcrystals

doi: 10.1038/srep11051

Figure Lengend Snippet: Silk gut powder samples (1 mg) from the BmFibH-polyhedrin/H1/FGF-2 and BmFibH-H1/FGF-2 lines suspended in PBS and diluted rhFGF-2 (10 ng) were placed on the inside filters of cell culture inserts. The samples in the cell culture inserts were air-dried and kept for one week at 25 °C (treated samples; column T). Untreated (column U) and treated samples in cell culture inserts were introduced into culture of serum-starved NIH3T3 cells. After 48 h of cultivation, cell counts were measured by WST-8 assay. Mean relative values of triplicate experiments are presented with standard deviations. * P < 0.01 vs . cells cultured with each sample of rhFGF-2 and silk gut powder from the BmFibH-H1/FGF-2 line after incubation for one week at 25 °C.

Article Snippet: Commercial recombinant human FGF-2 (rhFGF-2; R&D Biochemicals, Inc.) and H1/FGF-2-encapsulating polyhedra prepared from Sf21 cell culture were treated as above.

Techniques: Cell Culture, Incubation

Journal:

Article Title: In Vivo Modulation of FGF Biological Activity Alters Cranial Suture Fate

doi:

Figure Lengend Snippet: A: Representative Northern blot analysis demonstrating an increase in TGF-β1 mRNA expression in RNA isolated from vehicle and AdCALacZ-infected NRCs in response to a 6-hour stimulation with FGF-2 (10 ng/ml). This increase in TGF-β1 mRNA in response to FGF-2 was not seen in NRCs infected with AdCAFGF-TR. A similar pattern of TGF-β1 mRNA expression was seen in PFDCs after infection and stimulation (data not shown). 50 = 50 plaque forming units/cell; 100 = 100 plaque forming units/cell. B: Representative Northern blot analysis demonstrating an increase in collagen type I mRNA expression in RNA isolated from vehicle and AdCALacZ-infected PFDCs in response to 48-hour stimulation with FGF-2 (10 ng/ml). PFDCs infected with AdCAFGF-TR failed to demonstrate an increase in collagen type I mRNA. Interestingly, infected NRCs did not demonstrate differences in collagen type I gene expression in response to FGF-2 (data not shown). C: Cells infected with AdCAFGF-TR proliferated significantly slower than cells infected with vehicle and AdCALacZ. Graphic representation of BrdU incorporation by NRCs infected with vehicle, AdCALacZ, or AdCAFGF-TR. Note statistically significant decreased proliferation in NRCs infected with AdCAFGF-TR (*, P < 0.001). All cells were stimulated with rhFGF-2. D: Results of proliferation assay. Note significantly decreased proliferation of NRCs infected with AdCAFGF-TR (*, P < 0.001). All cells were stimulated with rhFGF-2. E: Representative Northern blot analysis demonstrating an increase in baseline TGF-β1 mRNA expression in RNA isolated from AdCAsFGF-2-infected NRCs. Similar pattern of TGF-β1 mRNA expression was seen in PFDCs after AdCAsFGF-2 infection (data not shown). F: Cells infected with AdCAsFGF-2 proliferated significantly faster than cells infected with vehicle and AdCALacZ. Graphic representation of BrdU incorporation by NRCs infected with vehicle, AdCALacZ, or AdCAsFGF-2. Note statistically significantly increased proliferation in NRCs infected with AdCAsFGF-2. (*, P < 0.001). G: Results of proliferation assay. Note significantly increased proliferation of NRCs infected with AdCAsFGF-2 (*, P < 0.001).

Article Snippet: After 24 hours (ie, 48 hours after infection), 10 ng/ml of recombinant human basic FGF (rhFGF-2; R&D Systems, Minneapolis, MN) was added to the serum-free media.

Techniques: Northern Blot, Expressing, Isolation, Infection, Gene Expression, BrdU Incorporation Assay, Proliferation Assay

Journal:

Article Title: In Vivo Modulation of FGF Biological Activity Alters Cranial Suture Fate

doi:

Figure Lengend Snippet: A: Northern (left) and Western blot (right) analysis demonstrating high-level expression of FGF-TR. Note increase in FGF-TR mRNA and protein with increasing plaque-forming units. Compare AdCAFGF-TR 50 with AdCAFGF-TR 100. 50 = 50 plaque-forming units/cell; 100 = 100 plaque-forming units/cell. B: Western blot analysis demonstrating increased phosphorylation of ERK-1 and -2 after FGF-2 stimulation (+) in vehicle and AdCALacZ-infected NRCs (top immunoblot). In contrast, AdCAFGF-TR-infected NRCs demonstrate no increase in ERK-1 and -2 phosphorylation in response to rhFGF-2 stimulation. Total ERK-2 immunoblot of same blot (bottom immunoblot) demonstrating presence of similar levels of unphosphorylated protein in all lanes. C: Western blot analysis for FGF-2 demonstrates increased expression of FGF-2 protein in NRCs infected with AdCsFGF-2. D: Western blot analysis demonstrating increased phosphorylation of ERK-1 and -2 after AdCAsFGF-2 infection of NRCs (top immunoblot). Total ERK-1 and -2 immunoblot of same membrane (bottom immunoblot) demonstrating presence of similar levels of unphosphorylated protein in all lanes. In this experiment, cells were maintained in serum-free media without the addition of rhFGF-2.

Article Snippet: After 24 hours (ie, 48 hours after infection), 10 ng/ml of recombinant human basic FGF (rhFGF-2; R&D Systems, Minneapolis, MN) was added to the serum-free media.

Techniques: Northern Blot, Western Blot, Expressing, Phospho-proteomics, Infection, Membrane