fibroblast supplemental growth factors  (ATCC)

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

doi: 10.1111/wrr.13177

Figure Lengend Snippet: FIGURE 1 Porcine-derived collagen peptides promotes a trend wise increase in FAK and ERK signalling but not Akt in wounded keratinocytes. (A) Schematic diagram illustrating collagen interaction with cell surface integrin receptors leads to downstream activation of signalling pathways associated with cellular proliferation. (B) Representative western blot for integrin α2 (130 kDa), integrin β1 (130 kDa), p-FAK (110 kDa), FAK (110 kDa), p-Akt (60 kDa), Akt (60 kDa), p-ERK (42–44 kDa), ERK (42–44 kDa) and GAPDH (37 kDa) expression in wounded keratinocytes taken at 0, 24, 48, and 72 h post-wounding in the presence or absence of 1 mg/mL porcine-derived collagen peptides (PCP). Densitometric expression of (C) integrin α2, (D) integrin β1, (E) p-ERK/ERK, (F) p-FAK/FAK or (G) p-Akt/Akt expression relative to GAPDH expression in wounded keratinocytes taken at 0, 24, 48, and 72 h post-wounding the presence or absence of 1 mg/mL PCP (Mean ± SD, N = 3 independent experiments using 3 different biological samples, Two-way ANOVA with Tukey's multiple comparisons test, ns).

Article Snippet: Primary human keratinocytes and dermal fibroblasts were isolated from surplus human skin following informed consent (REC reference 19/NE/004_Lovat) and maintained as previously described.12 Primary keratinocytes were cultured in either EpiLife media (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 1% human keratinocyte growth factor supplement (HKGS) (ThermoFisher Scientific, USA) and 1% penicillin–streptomycin-amphotericin (PSA) (Lonza, Verviers, Belgium), or Keratinocyte Growth Medium 2 (Promocell, Heidelberg, Germany) supplemented with SupplementMix (Promocell, Germany) and 1% PSA (Lonza, Belgium).

Techniques: Derivative Assay, Activation Assay, Western Blot, Expressing

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

doi: 10.1111/wrr.13177

Figure Lengend Snippet: FIGURE 2 Knockdown of integrin β1 significantly impairs porcine-derived collagen peptide-induced activation of ERK and FAK and wound closure of keratinocytes. (A) Representative western blot for integrin α2 (130 kDa), integrin β1 (130 kDa), p-FAK (110 kDa), FAK (110 kDa), p-Akt (60 kDa), Akt (60 kDa), p-ERK (42–44 kDa), ERK (42–44 kDa), and GAPDH (37 kDa) expression in wounded keratinocytes at 24, 48, and 72 h post-wounding in the presence or absence of 1 mg/mL porcine-derived collagen peptides (PCP) following treatment with either SiCtrl or ITGB1 siRNA. Densitometric expression of (B) integrin β1, (C) integrin α2, (D) p-ERK/ERK, (E) p-FAK/FAK expression relative to GAPDH expression in wounded keratinocytes at 24, 48, and 72 h post-wounding in the presence or absence of 1 mg/mL PCP following treatment with either SiCtrl or ITGB1 siRNA (Mean ± SD, N = 3 independent experiments using 3 different biological samples, Two-way ANOVA with Tukey's multiple comparisons test, *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001). (F) Schematic diagram illustrating potential downstream pathway activation following PCP binding to the integrin α2β1 receptor in order to promote keratinocyte wound closure. (G) Primary keratinocytes were seeded onto either uncoated wells or wells pre-coated with 1 mg/mL PCP before being treated with ITGB1 siRNA or siCtrl, scratch wound induction and monitoring of wound closure over 72 h (Mean ± SD, N = 3 independent experiments using three different biological samples, One-way ANOVA with Tukey's multiple comparisons test, *p<0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001).

Article Snippet: Primary human keratinocytes and dermal fibroblasts were isolated from surplus human skin following informed consent (REC reference 19/NE/004_Lovat) and maintained as previously described.12 Primary keratinocytes were cultured in either EpiLife media (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 1% human keratinocyte growth factor supplement (HKGS) (ThermoFisher Scientific, USA) and 1% penicillin–streptomycin-amphotericin (PSA) (Lonza, Verviers, Belgium), or Keratinocyte Growth Medium 2 (Promocell, Heidelberg, Germany) supplemented with SupplementMix (Promocell, Germany) and 1% PSA (Lonza, Belgium).

Techniques: Knockdown, Derivative Assay, Activation Assay, Western Blot, Expressing, Binding Assay

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

doi: 10.1111/wrr.13177

Figure Lengend Snippet: FIGURE 3 Porcine-derived collagen peptides increase expression of integrin α2, integrin α5 and integrin β1 in keratinocytes. Relative cell surface expression of (A) integrin β1, (B) integrin α2, (C) integrin α5, (E) integrin α3, and (E) integrin α6 compared to the control in unwounded primary keratinocytes treated in the presence or absence of 1 mg/mL porcine-derived collagen peptides (PCP) for 72 h before analysis using flow cytometry (Mean ± SD, N = 3 independent experiments using three different biological samples, Unpaired t-test, *p ≤0.05, **p ≤0.01).

Article Snippet: Primary human keratinocytes and dermal fibroblasts were isolated from surplus human skin following informed consent (REC reference 19/NE/004_Lovat) and maintained as previously described.12 Primary keratinocytes were cultured in either EpiLife media (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 1% human keratinocyte growth factor supplement (HKGS) (ThermoFisher Scientific, USA) and 1% penicillin–streptomycin-amphotericin (PSA) (Lonza, Verviers, Belgium), or Keratinocyte Growth Medium 2 (Promocell, Heidelberg, Germany) supplemented with SupplementMix (Promocell, Germany) and 1% PSA (Lonza, Belgium).

Techniques: Derivative Assay, Expressing, Control, Flow Cytometry

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

doi: 10.1111/wrr.13177

Figure Lengend Snippet: FIGURE 4 Inhibition of integrin α2β1 impairs keratinocyte adhesion to PCP. (A) Relative cell count of keratinocytes adhered to either collagen I or PCP-coated wells in the presence or absence of 10 μg/mL integrin α2β1 blocking antibody after 2 h (Mean ± SD, N = 3 independent experiments using three different biological samples, one-way ANOVA with Tukey's multiple comparisons test, *p <0.05, **p <0.01, ***p <0.001). (B) Relative cell count of keratinocytes adhered to either fibronectin or PCP-coated wells in the presence or absence of 10 μg/mL integrin α5β1blocking antibody after 2 h (Mean ± SD, N = 3 independent experiments using three different biological samples, one-way ANOVA with Tukey's multiple comparisons test, *p <0.05).

Article Snippet: Primary human keratinocytes and dermal fibroblasts were isolated from surplus human skin following informed consent (REC reference 19/NE/004_Lovat) and maintained as previously described.12 Primary keratinocytes were cultured in either EpiLife media (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 1% human keratinocyte growth factor supplement (HKGS) (ThermoFisher Scientific, USA) and 1% penicillin–streptomycin-amphotericin (PSA) (Lonza, Verviers, Belgium), or Keratinocyte Growth Medium 2 (Promocell, Heidelberg, Germany) supplemented with SupplementMix (Promocell, Germany) and 1% PSA (Lonza, Belgium).

Techniques: Inhibition, Cell Counting, Blocking Assay