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

Journal: Developmental biology

Article Title: A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice

doi: 10.1016/j.ydbio.2018.06.017

Figure Lengend Snippet: A. Brightfield (right panels) and Venus-fluorescence (middle) in Spry4H2B-Venus ESCs grown in serum/LIF medium (upper panels) or serum/LIF supplemented with 1 μM PD0325901 for 3 days (lower panels). Scale bar, 25 μm. B. Schematic diagram depicting experimental approach to determine ERK responsiveness of the reporter. Cells were cultured for 3 days in serum/LIF conditions supplemented with 1 μM PD0325901 to downregulate reporter expression, followed by transfer into subsaturating PD03 concentrations for one day. C. Flow cytometry analysis of Venus expression in Spry4H2B-Venus ESCs treated as indicated in (B). Uppermost histogram is from the Spry4 wild-type parental cell line. D. Quantification of results from (C). Expression of Venus in cells maintained in serum/LIF medium was normalized to 1, data points show average +/− SD from four independent experiments. E. Schematic of experimental approach to determine FGF responsiveness of the reporter. Wild-type and Fgf4 mutant cells were cultured in 2i/LIF conditions for three days followed by transfer into N2B27 medium supplemented with various doses of FGF4 or FGF2 for one day, or no FGF for parental, wild-type and mutant controls. F. Flow cytometry analysis of Venus expression in Fgf4 wild-type (wt) and Fgf4−/− mutant Spry4H2B-Venus ESCs treated as indicated in (E). The level of Venus expression in Fgf4 mutant cells is comparable to wt parental ESCs without the reporter, indicating that reporter expression in Fgf4 wt cells is mainly triggered by ESC-secreted FGF4. N = 3, one representative experiment shown. G. Total ERK and ppERK levels in Fgf4 mutant cells treated as indicated in (E) determined by immunoblotting. N = 3, one representative experiment shown. H. Relationship between ERK phosphorylation and Venus expression in Fgf4−/− mutant ESCs treated with different doses of FGF4 and FGF2 compiled from data in panels F and G. Data normalized to ppERK and Venus levels in cells treated with the highest dose of FGF4 (shown as 1). Data points indicate average +/− SD from two (Fgf4 mutant, 0 ng/ml FGF) or three (all other data points) independent experiments.

Article Snippet: For activation of the FGF/ERK pathway in ESCs, 10–100 ng/ml of recombinant human FGF2 (rhFGF2, CellGS) or FGF4 (rhFGF4, Peprotech) was added to the specified culture medium with 1 μg/ml of Heparin Sulfate (Sigma).

Techniques: Fluorescence, Cell Culture, Expressing, Flow Cytometry, Mutagenesis, Western Blot, Phospho-proteomics

Journal: Cell stem cell

Article Title: Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling

doi: 10.1016/j.stem.2017.03.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For organoid experiments, cells were cultured in “CFK media” containing 3 μM CHIR99021, 10 ng/mL rhFGF10, and 10 ng/mL rhKGF or “2+10 media” containing 250 ng/mL recombinant human FGF2 (rhFGF2, R&D Systems) and 100 ng/mL rhFGF10.

Techniques: Marker, Recombinant, Lysis, Microarray, Plasmid Preparation, Sequencing, Gene Expression, Software, Gentle, Transfection