rhfgf-7  (FUJIFILM)

Journal: FASEB BioAdvances

Article Title: Effects of transgenic silk materials that incorporate FGF‐7 protein microcrystals on the proliferation and differentiation of human keratinocytes

doi: 10.1096/fba.2020-00078

Figure Lengend Snippet: Generation of transgenic silkworms that express H1/FGF‐7 in the posterior silk gland. A ) Schematic representation of the plasmid vector used to generate transgenic silkworms. The plasmid vector pBacMCS[UAS‐H1/FGF‐7‐SV40/3xP3‐EGFP] encoding H1/FGF‐7 was used to generate the transgenic line termed UAS‐H1/FGF‐7. The piggyBac right and left inverted terminal repeats (ITRs) (L and R) are indicated by arrows. B ) Generation of the FH‐H1/FGF‐7 line. The FH‐GAL4 line ²⁹ was mated with the UAS‐H1/FGF‐7 line to generate the FH‐H1/FGF‐7 line in which expression of EGFP and discosome sp. Red (DsRed) as genetic markers is visible in the eyes. C ) Generation of the FH‐poly/H1/FGF‐7 line. The FH‐polyhedrin line was mated with the UAS‐H1/FGF‐7 line to generate the FH‐poly/H1/FGF‐7 line with the expression of EGFP and DsRed visible in the eyes and kynurenine 3‐monooxygenase (KMO) specific expression in the skin, ⁷ as genetic markers.

Article Snippet: Recombinant human FGF‐7 (rhFGF‐7; 100 ng/ml; Wako Chemical) was added as a positive control.

Techniques: Transgenic Assay, Plasmid Preparation, Expressing

Journal: FASEB BioAdvances

Article Title: Effects of transgenic silk materials that incorporate FGF‐7 protein microcrystals on the proliferation and differentiation of human keratinocytes

doi: 10.1096/fba.2020-00078

Figure Lengend Snippet: Immunoblotting analysis of H1/FGF‐7 expressed in posterior silk glands. Protein samples from 50,000 empty polyhedra cubes (negative control; lane 1) and H1/FGF‐7‐polyhedra (positive control; lane 2) produced in baculovirus‐infected Sf21 cells, in addition to proteins immunoprecipitated from 50 mg PSGs from w1 ‐ pnd (lane 3) and FH‐H1/FGF‐7 larvae (lane 4) were electrophoresed on 12.5% SDS‐PAGE and analyzed by immunoblotting with anti‐FGF‐7 antibodies. Protein size markers are indicated on the left

Article Snippet: Recombinant human FGF‐7 (rhFGF‐7; 100 ng/ml; Wako Chemical) was added as a positive control.

Techniques: Western Blot, Negative Control, Positive Control, Produced, Infection, Immunoprecipitation, SDS Page

Journal: FASEB BioAdvances

Article Title: Effects of transgenic silk materials that incorporate FGF‐7 protein microcrystals on the proliferation and differentiation of human keratinocytes

doi: 10.1096/fba.2020-00078

Figure Lengend Snippet: Detection of H1/FGF‐7 associated with polyhedra. A, Detection of H1/FGF‐7 in polyhedra using immunoblotting. Polyhedra (50,000 cubes) collected from PSGs of FH‐polyhedrin (lane 1, ref. 7) and FH‐poly/H1/FGF‐7 larvae (lane 2) were analyzed using immunoblotting with anti‐FGF‐7 antibodies. B, Confocal microscopy analysis of H1/FGF‐7 and polyhedrin expression on polyhedra by immunofluorescence. Polyhedra collected from PSGs of FH‐poly/H1/FGF‐7 larvae were fixed on a glass‐base dish and examined using immunofluorescence with anti‐FGF‐7 antibody and anti‐polyhedrin antibody. Scale bars, 10 µm

Article Snippet: Recombinant human FGF‐7 (rhFGF‐7; 100 ng/ml; Wako Chemical) was added as a positive control.

Techniques: Western Blot, Confocal Microscopy, Expressing, Immunofluorescence

Journal: FASEB BioAdvances

Article Title: Effects of transgenic silk materials that incorporate FGF‐7 protein microcrystals on the proliferation and differentiation of human keratinocytes

doi: 10.1096/fba.2020-00078

Figure Lengend Snippet: Release of H1/FGF‐7 from posterior silk gland powders (PSGPs) incorporating FGF‐7‐polyhedra. The amount of H1/FGF‐7 released from A, H1/FGF‐7‐polyhedra (5.0 × 10 5 cubes) or B, Posterior silk gland powders (1.0 mg) from FH‐poly/H1/FGF‐7 larvae were examined using an enzyme‐linked immunosorbent assay (ELISA) with anti‐FGF‐7 antibody. Each sample was added into keratinocyte‐conditioned media using the filter of a cell culture insert (see MATERIALS AND METHODS) and incubated for 14 days. The medium was changed and collected on days 3, 7, 10, and 14. The amount of FGF‐7 in the collected culture medium was determined using an ELISA. Data shown are the means ±standard deviation (SD) of triplicate assays (N = 3 independent samples)

Article Snippet: Recombinant human FGF‐7 (rhFGF‐7; 100 ng/ml; Wako Chemical) was added as a positive control.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Standard Deviation

Journal: FASEB BioAdvances

Article Title: Effects of transgenic silk materials that incorporate FGF‐7 protein microcrystals on the proliferation and differentiation of human keratinocytes

doi: 10.1096/fba.2020-00078

Figure Lengend Snippet: Proliferation of NHEK induced by silk gland powders (SGPs) containing H1/FGF‐7‐polyhedra. A, Phosphorylation of p44/42 mitogen‐activated protein kinase (MAPK) in cultured NHEKs. NHEKs were supplement‐starved overnight and left untreated as a negative control (no SGPs; lane 1) or treated with 10 mg of MSGPs from S1‐polyhedrin (lane 2), S1‐H1/FGF‐7 (lane 4) or S1‐poly/H1/FGF‐7 larvae (lane 5), or 10 mg of PSGPs from FH‐polyhedrin (lane 3), FH‐H1/FGF‐7 (lane 6), or FH‐poly/H1/FGF‐7 larvae (lane 7) for 1 h. NHEKs were also treated with 100 ng/ml rhFGF‐7 (lane 8) as a positive control. The NHEK lysates were examined using immunoblotting with antibodies against either p44/p42 MAPK or phosphorylated p44/p42 MAPK. B, Keratinocyte proliferation. Relative numbers of living cells were determined using a WST‐8 assay. NHEKs were cultured in media containing rhFGF‐7 (1 ng, 2 ng or 5 ng) or cultured with cell culture inserts with middle silk gland powders from S1‐polyhedrin (500 µg), S1‐H1/FGF‐7 (50, 250, or 500 µg) or S1‐poly/H1/FGF‐7 larvae (50, 250, or 500 µg), or posterior silk gland powders from FH‐polyhedrin (200 µg), FH‐H1/FGF‐7 (50, 100, or 200 µg) or FH‐poly/H1/FGF‐7 larvae (50, 100, or 200 µg). After incubation for 72 h, cell numbers were analyzed using a WST‐8 assay. Absorbance at 450 nm was determined using a microplate reader. Data are the mean ±SD of triplicate assays. * p < 0.05, ** p < 0.01

Article Snippet: Recombinant human FGF‐7 (rhFGF‐7; 100 ng/ml; Wako Chemical) was added as a positive control.

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

Journal: FASEB BioAdvances

Article Title: Effects of transgenic silk materials that incorporate FGF‐7 protein microcrystals on the proliferation and differentiation of human keratinocytes

doi: 10.1096/fba.2020-00078

Figure Lengend Snippet: 3D keratinocyte cultures. A, Hematoxylin and eosin (HE) staining in a 3D culture of keratinocytes. NHEKs were cultured on a collagen gel containing 800 µg posterior silk gland powders (PSGPs) from FH‐polyhedrin or FH‐poly/H1/FGF‐7 larvae in the basal medium for 2 days and in a differentiation medium (basal medium containing 1.2 mM Ca 2+ ) for 14 days. The NHEKs were cultured in the same way on collagen with 100 ng/ml rhFGF‐7 in basal and differentiation media as a control. After cultivation for 14 days in differentiation media, 3D cultures were sectioned, subjected to HE staining and observed under a microscope. Scale bar, 50 µm. B, Immunohistochemical staining of keratinocyte 3D cultures. Immunohistochemical staining of keratin 10 (K10), keratin 14 (K14), Loricrin (Lor), and Filaggrin (Fil) in 3D cultures of keratinocytes incubated with PSGPs from FH‐poly/H1/FGF‐7 larvae. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole. Scale bar, 50 µm

Article Snippet: Recombinant human FGF‐7 (rhFGF‐7; 100 ng/ml; Wako Chemical) was added as a positive control.

Techniques: Staining, Cell Culture, Microscopy, Immunohistochemical staining, Incubation