keratinocyte-sfm medium Search Results


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  • 97
    Lonza serum free keratinocyte basal medium kbm
    Serum Free Keratinocyte Basal Medium Kbm, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
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    95
    Thermo Fisher keratinocyte sfm
    Effects of KGF infection on hESCs proliferation. hESCs were cultured in a <t>keratinocyte</t> serum-free medium and transfected with AdKGF or AdGFP. After further culture for 12 h, the medium was changed, and after an additional 48 h in culture, cell growth
    Keratinocyte Sfm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher keratinocyte sfm medium
    Effects of KGF infection on hESCs proliferation. hESCs were cultured in a <t>keratinocyte</t> serum-free medium and transfected with AdKGF or AdGFP. After further culture for 12 h, the medium was changed, and after an additional 48 h in culture, cell growth
    Keratinocyte Sfm Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratinocyte sfm medium/product/Thermo Fisher
    Average 99 stars, based on 1148 article reviews
    Price from $9.99 to $1999.99
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    91
    Thermo Fisher keratinocyte serum free medium ksfm
    Serum and Ca ++ affect growth and TGFβ-induced hypermotility differently a,b. Effects of [Ca ++ ] and serum on <t>keratinocyte</t> and prostate epithelial cell growth. Panel a: G5Ep and HPrE-1 cells plated at low density (~250 cells/cm 2 ) in <t>Ksfm</t> supplemented with 0.1, 0.4, or 1.0 mM Ca ++ or with 0.4 mM Ca ++ and 1% calf serum. Note that both cell types grew as well or better in higher [Ca ++ ] but were strongly inhibited by addition of 1% serum to the medium. Panel b: typical fields of HPrE-1 cells in the various conditions. Note inability to form closely packed colonies in low and medium [Ca ++ ] and abnormal elongation associated with growth inhibition in presence of serum. c. Increased sensitivity to TGFβ growth inhibition of keratinocyte and prostate epithelial cells when plated on serum-precoated dishes. Cells were plated in Ksfm in wells that were not pretreated or that were precoated with 10% serum. Note little change in growth rate −TGFβ and a significant dose-response shift toward increased TGFβ-induced growth inhibition for both cell types on serum-precoated dishes. d. A substratum-bound component of serum blocks TGFβ-induced hypermotility. G5Ep and HPrE-1 cells were plated + or −TGFβ in Ksfm with 0.2 mM Ca ++ in untreated or serum-precoated wells. Note lack of hypermotility response to TGFβ of cells in serum-precoated wells. e. TGFβ-induced hypermotility is inversely related to [Ca ++ ] of the medium. G5Ep cells were plated + or −TGFβ or on Lam332′-precoated surfaces in Ksfm with [Ca ++ ] = 0.1 mM, 0.4 mM, or 1.0 mM. Note that hypermotility induced by TGFβ was greatly reduced, and that by Lam332′ slightly attenuated, by increasing [Ca ++ ].
    Keratinocyte Serum Free Medium Ksfm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 685 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 685 article reviews
    Price from $9.99 to $1999.99
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    95
    Millipore keratinocyte serum free medium
    Serum and Ca ++ affect growth and TGFβ-induced hypermotility differently a,b. Effects of [Ca ++ ] and serum on <t>keratinocyte</t> and prostate epithelial cell growth. Panel a: G5Ep and HPrE-1 cells plated at low density (~250 cells/cm 2 ) in <t>Ksfm</t> supplemented with 0.1, 0.4, or 1.0 mM Ca ++ or with 0.4 mM Ca ++ and 1% calf serum. Note that both cell types grew as well or better in higher [Ca ++ ] but were strongly inhibited by addition of 1% serum to the medium. Panel b: typical fields of HPrE-1 cells in the various conditions. Note inability to form closely packed colonies in low and medium [Ca ++ ] and abnormal elongation associated with growth inhibition in presence of serum. c. Increased sensitivity to TGFβ growth inhibition of keratinocyte and prostate epithelial cells when plated on serum-precoated dishes. Cells were plated in Ksfm in wells that were not pretreated or that were precoated with 10% serum. Note little change in growth rate −TGFβ and a significant dose-response shift toward increased TGFβ-induced growth inhibition for both cell types on serum-precoated dishes. d. A substratum-bound component of serum blocks TGFβ-induced hypermotility. G5Ep and HPrE-1 cells were plated + or −TGFβ in Ksfm with 0.2 mM Ca ++ in untreated or serum-precoated wells. Note lack of hypermotility response to TGFβ of cells in serum-precoated wells. e. TGFβ-induced hypermotility is inversely related to [Ca ++ ] of the medium. G5Ep cells were plated + or −TGFβ or on Lam332′-precoated surfaces in Ksfm with [Ca ++ ] = 0.1 mM, 0.4 mM, or 1.0 mM. Note that hypermotility induced by TGFβ was greatly reduced, and that by Lam332′ slightly attenuated, by increasing [Ca ++ ].
    Keratinocyte Serum Free Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratinocyte serum free medium/product/Millipore
    Average 95 stars, based on 46 article reviews
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    keratinocyte serum free medium - by Bioz Stars, 2020-10
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    90
    Thermo Fisher keratinocyte serum free ksf medium
    Serum and Ca ++ affect growth and TGFβ-induced hypermotility differently a,b. Effects of [Ca ++ ] and serum on <t>keratinocyte</t> and prostate epithelial cell growth. Panel a: G5Ep and HPrE-1 cells plated at low density (~250 cells/cm 2 ) in <t>Ksfm</t> supplemented with 0.1, 0.4, or 1.0 mM Ca ++ or with 0.4 mM Ca ++ and 1% calf serum. Note that both cell types grew as well or better in higher [Ca ++ ] but were strongly inhibited by addition of 1% serum to the medium. Panel b: typical fields of HPrE-1 cells in the various conditions. Note inability to form closely packed colonies in low and medium [Ca ++ ] and abnormal elongation associated with growth inhibition in presence of serum. c. Increased sensitivity to TGFβ growth inhibition of keratinocyte and prostate epithelial cells when plated on serum-precoated dishes. Cells were plated in Ksfm in wells that were not pretreated or that were precoated with 10% serum. Note little change in growth rate −TGFβ and a significant dose-response shift toward increased TGFβ-induced growth inhibition for both cell types on serum-precoated dishes. d. A substratum-bound component of serum blocks TGFβ-induced hypermotility. G5Ep and HPrE-1 cells were plated + or −TGFβ in Ksfm with 0.2 mM Ca ++ in untreated or serum-precoated wells. Note lack of hypermotility response to TGFβ of cells in serum-precoated wells. e. TGFβ-induced hypermotility is inversely related to [Ca ++ ] of the medium. G5Ep cells were plated + or −TGFβ or on Lam332′-precoated surfaces in Ksfm with [Ca ++ ] = 0.1 mM, 0.4 mM, or 1.0 mM. Note that hypermotility induced by TGFβ was greatly reduced, and that by Lam332′ slightly attenuated, by increasing [Ca ++ ].
    Keratinocyte Serum Free Ksf Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratinocyte serum free ksf medium/product/Thermo Fisher
    Average 90 stars, based on 132 article reviews
    Price from $9.99 to $1999.99
    keratinocyte serum free ksf medium - by Bioz Stars, 2020-10
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    91
    ScienCell serum free keratinocyte medium
    Serum and Ca ++ affect growth and TGFβ-induced hypermotility differently a,b. Effects of [Ca ++ ] and serum on <t>keratinocyte</t> and prostate epithelial cell growth. Panel a: G5Ep and HPrE-1 cells plated at low density (~250 cells/cm 2 ) in <t>Ksfm</t> supplemented with 0.1, 0.4, or 1.0 mM Ca ++ or with 0.4 mM Ca ++ and 1% calf serum. Note that both cell types grew as well or better in higher [Ca ++ ] but were strongly inhibited by addition of 1% serum to the medium. Panel b: typical fields of HPrE-1 cells in the various conditions. Note inability to form closely packed colonies in low and medium [Ca ++ ] and abnormal elongation associated with growth inhibition in presence of serum. c. Increased sensitivity to TGFβ growth inhibition of keratinocyte and prostate epithelial cells when plated on serum-precoated dishes. Cells were plated in Ksfm in wells that were not pretreated or that were precoated with 10% serum. Note little change in growth rate −TGFβ and a significant dose-response shift toward increased TGFβ-induced growth inhibition for both cell types on serum-precoated dishes. d. A substratum-bound component of serum blocks TGFβ-induced hypermotility. G5Ep and HPrE-1 cells were plated + or −TGFβ in Ksfm with 0.2 mM Ca ++ in untreated or serum-precoated wells. Note lack of hypermotility response to TGFβ of cells in serum-precoated wells. e. TGFβ-induced hypermotility is inversely related to [Ca ++ ] of the medium. G5Ep cells were plated + or −TGFβ or on Lam332′-precoated surfaces in Ksfm with [Ca ++ ] = 0.1 mM, 0.4 mM, or 1.0 mM. Note that hypermotility induced by TGFβ was greatly reduced, and that by Lam332′ slightly attenuated, by increasing [Ca ++ ].
    Serum Free Keratinocyte Medium, supplied by ScienCell, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/serum free keratinocyte medium/product/ScienCell
    Average 91 stars, based on 16 article reviews
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    91
    Cambrex serum free keratinocyte growth medium
    The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human <t>keratinocytes</t>
    Serum Free Keratinocyte Growth Medium, supplied by Cambrex, used in various techniques. Bioz Stars score: 91/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher keratinocyte serum free medium dk sfm
    The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human <t>keratinocytes</t>
    Keratinocyte Serum Free Medium Dk Sfm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 140 article reviews
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    95
    Millipore serum free growth medium
    The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human <t>keratinocytes</t>
    Serum Free Growth Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    DS Pharma Biomedical human keratinocyte serum free medium
    An EMT-like phenotype was observed in the GnT-V Tg mouse <t>keratinocytes.</t> The EMT-associated transcriptional factors snail, twist, E-cadherin, and, N-cadherin were evaluated by quantitative RT-PCR, and target gene expression was normalized to GAPDH. Results
    Human Keratinocyte Serum Free Medium, supplied by DS Pharma Biomedical, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of KGF infection on hESCs proliferation. hESCs were cultured in a keratinocyte serum-free medium and transfected with AdKGF or AdGFP. After further culture for 12 h, the medium was changed, and after an additional 48 h in culture, cell growth

    Journal: Molecules and Cells

    Article Title: The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

    doi: 10.1007/s10059-013-0093-y

    Figure Lengend Snippet: Effects of KGF infection on hESCs proliferation. hESCs were cultured in a keratinocyte serum-free medium and transfected with AdKGF or AdGFP. After further culture for 12 h, the medium was changed, and after an additional 48 h in culture, cell growth

    Article Snippet: Then the cells were centrifuged at 1,200 rpm for 5 min, resuspended in a keratinocyte serum-free medium (K-SFM) (supplemented with 5 ng/ml epidermal growth factor and 50 mg/ml bovine pituitary extract; Gibco), and plated into 25-cm2 culture flasks preprocessed using type IV collagen (100 μg/ml; Sigma, USA).

    Techniques: Infection, Cell Culture, Transfection

    1 mM 4-PBA treatment results in reduced keratinocyte migration. A Two-dimensional wound closure assays were performed using Ibidi μ-dishes with inserts. Wounds at 0, 3, 6, and 9 h are shown. Note the delayed wound closure in NHK cells compared to KRT14mut and KRT5mut cells. After 4-PBA treatment all wounds show delayed closure. B The graph shows the quantification of the wound area after 3, 6, and 9 h as percentage of the initial area. These experiments were done in triplicates and 2 different keratinocyte cell lines were used for each group. KRT14mut and KRT5mut cells migrate significantly quicker than NHK [for both ***P

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: 1 mM 4-PBA treatment results in reduced keratinocyte migration. A Two-dimensional wound closure assays were performed using Ibidi μ-dishes with inserts. Wounds at 0, 3, 6, and 9 h are shown. Note the delayed wound closure in NHK cells compared to KRT14mut and KRT5mut cells. After 4-PBA treatment all wounds show delayed closure. B The graph shows the quantification of the wound area after 3, 6, and 9 h as percentage of the initial area. These experiments were done in triplicates and 2 different keratinocyte cell lines were used for each group. KRT14mut and KRT5mut cells migrate significantly quicker than NHK [for both ***P

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Migration

    Skin equivalents with NHK and EBS cells show several intra- and subepidermal splits after 1 mM 4-PBA treatment, as well as perturbed staining patterns of laminin-332 and of integrin β4. A Skin equivalents with control vs EBS keratinocytes for the epidermal part and control normal human fibroblasts for the dermal part were employed to assess the effect of 4-PBA in a 3D model. Only a few areas of dermo-epidermal detachment are present in the EBS skin equivalents compared to the control skin equivalents, as shown with haematoxylin/ eosin staining. In the 4-PBA treated skin, however, several intra- and subepidermal splits (black arrows) were found, corroborating the adhesion defect. Scale: 100 μm. B Laminin-332 deposition was irregular and patchy in the NHK, whereas it also stained the suprabasal keratinocytes in the EBS cells. In the 4-PBA treated skin, again, several intra- and subepidermal splits (yellow arrows) were observed. Scale: 100 μm. C Integrin β4 showed also an irregular and more pronounced, patchy, suprabasal expression in the treated skin equivalents. Thus, 4-PBA treatment perturbs laminin-332 organization, which in turn seems to abrogate strictly polarized expression of integrin α6β4 in basal keratinocytes. The yellow arrows depict intra- and subepidermal splits, present mostly in the 4-PBA treated skin. Scale: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: Skin equivalents with NHK and EBS cells show several intra- and subepidermal splits after 1 mM 4-PBA treatment, as well as perturbed staining patterns of laminin-332 and of integrin β4. A Skin equivalents with control vs EBS keratinocytes for the epidermal part and control normal human fibroblasts for the dermal part were employed to assess the effect of 4-PBA in a 3D model. Only a few areas of dermo-epidermal detachment are present in the EBS skin equivalents compared to the control skin equivalents, as shown with haematoxylin/ eosin staining. In the 4-PBA treated skin, however, several intra- and subepidermal splits (black arrows) were found, corroborating the adhesion defect. Scale: 100 μm. B Laminin-332 deposition was irregular and patchy in the NHK, whereas it also stained the suprabasal keratinocytes in the EBS cells. In the 4-PBA treated skin, again, several intra- and subepidermal splits (yellow arrows) were observed. Scale: 100 μm. C Integrin β4 showed also an irregular and more pronounced, patchy, suprabasal expression in the treated skin equivalents. Thus, 4-PBA treatment perturbs laminin-332 organization, which in turn seems to abrogate strictly polarized expression of integrin α6β4 in basal keratinocytes. The yellow arrows depict intra- and subepidermal splits, present mostly in the 4-PBA treated skin. Scale: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Staining, Expressing

    TNFα is upregulated after 4-PBA treatment on protein and mRNA level. A TNFα abundance was evaluated by dot blots in NHK and EBS untreated and 4-PBA-treated cells. TNFα appeared to be upregulated after 4-PBA treatment. Paired t-test was used for quantification [NHK vs NHK+ * P = .025, NHK vs KRT14mut ** P = .0011, KRT14mut vs KRT14mut + P = .3512, KRT5mut vs KRT5mut + ** P = .0086 and NHK vs KRT5mut *** P = .0003]. B Quantitative real-time PCR (qPCR) analysis of RNA isolated from keratinocyte cell lines before and after 1 mM 4-PBA treatment for TNFα. Expression of TNFα normalized to the expression of GAPDH is shown as the percentage of expression in untreated cells. 4-PBA treatment upregulated the expression of all four genes, but significantly only in the NHK. Paired t-test was used [NHK vs NHK+ * P = .0194, KRT14mut vs KRT14mut + P = .2364 and KRT5mut vs KRT5mut + P = .2174].

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: TNFα is upregulated after 4-PBA treatment on protein and mRNA level. A TNFα abundance was evaluated by dot blots in NHK and EBS untreated and 4-PBA-treated cells. TNFα appeared to be upregulated after 4-PBA treatment. Paired t-test was used for quantification [NHK vs NHK+ * P = .025, NHK vs KRT14mut ** P = .0011, KRT14mut vs KRT14mut + P = .3512, KRT5mut vs KRT5mut + ** P = .0086 and NHK vs KRT5mut *** P = .0003]. B Quantitative real-time PCR (qPCR) analysis of RNA isolated from keratinocyte cell lines before and after 1 mM 4-PBA treatment for TNFα. Expression of TNFα normalized to the expression of GAPDH is shown as the percentage of expression in untreated cells. 4-PBA treatment upregulated the expression of all four genes, but significantly only in the NHK. Paired t-test was used [NHK vs NHK+ * P = .0194, KRT14mut vs KRT14mut + P = .2364 and KRT5mut vs KRT5mut + P = .2174].

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Real-time Polymerase Chain Reaction, Isolation, Expressing

    Keratin aggregates in EBS keratinocytes at resting state and after stress: rescue by 4-PBA. A Analysis of the subcellular localisation of plectin and keratin 5 as shown by immunofluorescence staining of control normal human keratinocytes (NHK), keratinocytes from patients with mutations in the keratin 14 gene (KRT14mut) and keratinocytes from patients with mutations in the keratin 5 gene (KRT5mut) with anti-plectin [Alexa555 (Cy3; red)] and keratin 5 [Alexa488 (GFP; green)] antibodies. Yellow arrows depict cells with aggregates of their keratin IF network. Scale bar: 50 μm. B The upper panel shows quantification of the keratinocytes with aggregates. The NHKs show no aggregates at resting state, whereas they are significantly more in KRT14mut than KRT5mut cells (for NHK versus KRT14mut ***P: 0.0001, for NHK versus KRT5mut *P: 0.0211 and for KRT5mut versus KRT14mut ***P: 0.0004, all done with student's t -test). In the lower panel the percentage of cells before (NHK, KRT14mut, KRT5mut) and after use of 4-PBA (NHK+, KRT14mut+, KRT5mut+) is shown. The data are presented as mean ± SEM. Intermediate filament (IF) aggregates are significantly less in cells treated with 1 mM 4-PBA [KRT14mut (*** P

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: Keratin aggregates in EBS keratinocytes at resting state and after stress: rescue by 4-PBA. A Analysis of the subcellular localisation of plectin and keratin 5 as shown by immunofluorescence staining of control normal human keratinocytes (NHK), keratinocytes from patients with mutations in the keratin 14 gene (KRT14mut) and keratinocytes from patients with mutations in the keratin 5 gene (KRT5mut) with anti-plectin [Alexa555 (Cy3; red)] and keratin 5 [Alexa488 (GFP; green)] antibodies. Yellow arrows depict cells with aggregates of their keratin IF network. Scale bar: 50 μm. B The upper panel shows quantification of the keratinocytes with aggregates. The NHKs show no aggregates at resting state, whereas they are significantly more in KRT14mut than KRT5mut cells (for NHK versus KRT14mut ***P: 0.0001, for NHK versus KRT5mut *P: 0.0211 and for KRT5mut versus KRT14mut ***P: 0.0004, all done with student's t -test). In the lower panel the percentage of cells before (NHK, KRT14mut, KRT5mut) and after use of 4-PBA (NHK+, KRT14mut+, KRT5mut+) is shown. The data are presented as mean ± SEM. Intermediate filament (IF) aggregates are significantly less in cells treated with 1 mM 4-PBA [KRT14mut (*** P

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Immunofluorescence, Staining

    Cell-ECM interactions are disturbed with enhanced focal adhesions after 1 mM 4-PBA treatment in NHK and EBS cells. A For the keratinocyte adhesion assay, the cells were coated on collagen I or left on untreated plates. We refer to the adhesion of the untreated cells on collagen I as 100% adhesion. After 4-PBA treatment, the adhesion of NHK appeared unchanged, whereas significant effects were found in 4-PBA-treated KRT14mut and less in KRT5mut cells [for KRT14mut + * P = .0411 and for KRT5mut + P = .3267, student's t-test]. B Reduction of the fraction of adherent cells in NHK and EBS cells in the spinning disk assay after treatment with 1 mM 4-PBA [NHK+ P = .4296; KRT14mut + P = .0652; KRT5mut + P = .6818]. The effect of 4-PBA on adhesiveness is significant, with NHK and EBS cells taken together (All cells vs all cells+: * P = .0001, student's t-test). C The focal adhesion-associated molecule integrin β1 (green) was stained and found greatly enhanced after 1 mM 4-PBA treatment especially in control and KRT5-mutated cells. Scale: 200 μm. D 4-PBA treatment resulted in altered staining of the epidermal F-actin cytoskeleton in control and EBS keratinocytes, whereas the total amount of F-actin was unchanged, as shown by immunoblotting, suggesting altered distribution of the actin filaments in the cells [for KRT14mut + P = .9092 and for KRT5mut + P = .1796, student's t -test]. Scale bar: 50 μm. The IF stainings were done in triplicates with at least 2 different cell lines for each group, before and after 4-PBA treatment. Shown are representative results from one experiment. E Immunoblotting for laminin-332 revealed increased amounts in cell lysates after 1 mM 4-PBA treatment. Bar graphs on the right show expression of laminin-332 normalized to tubulin as percentage of expression in untreated cells. Values represent mean ± S.E.M., paired t-test was used; for NHK P = .6097, for KRT14mut P = .4084 and for KRT5mut P = .6291. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: Cell-ECM interactions are disturbed with enhanced focal adhesions after 1 mM 4-PBA treatment in NHK and EBS cells. A For the keratinocyte adhesion assay, the cells were coated on collagen I or left on untreated plates. We refer to the adhesion of the untreated cells on collagen I as 100% adhesion. After 4-PBA treatment, the adhesion of NHK appeared unchanged, whereas significant effects were found in 4-PBA-treated KRT14mut and less in KRT5mut cells [for KRT14mut + * P = .0411 and for KRT5mut + P = .3267, student's t-test]. B Reduction of the fraction of adherent cells in NHK and EBS cells in the spinning disk assay after treatment with 1 mM 4-PBA [NHK+ P = .4296; KRT14mut + P = .0652; KRT5mut + P = .6818]. The effect of 4-PBA on adhesiveness is significant, with NHK and EBS cells taken together (All cells vs all cells+: * P = .0001, student's t-test). C The focal adhesion-associated molecule integrin β1 (green) was stained and found greatly enhanced after 1 mM 4-PBA treatment especially in control and KRT5-mutated cells. Scale: 200 μm. D 4-PBA treatment resulted in altered staining of the epidermal F-actin cytoskeleton in control and EBS keratinocytes, whereas the total amount of F-actin was unchanged, as shown by immunoblotting, suggesting altered distribution of the actin filaments in the cells [for KRT14mut + P = .9092 and for KRT5mut + P = .1796, student's t -test]. Scale bar: 50 μm. The IF stainings were done in triplicates with at least 2 different cell lines for each group, before and after 4-PBA treatment. Shown are representative results from one experiment. E Immunoblotting for laminin-332 revealed increased amounts in cell lysates after 1 mM 4-PBA treatment. Bar graphs on the right show expression of laminin-332 normalized to tubulin as percentage of expression in untreated cells. Values represent mean ± S.E.M., paired t-test was used; for NHK P = .6097, for KRT14mut P = .4084 and for KRT5mut P = .6291. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Cell Adhesion Assay, Staining, Expressing

    Effects of 1 mM 4-PBA on cell apoptosis and IL1β expression. A. Colony-forming efficiency assay of 4 different primary NHKs, treated with different dosages of 4-PBA for 1 week is shown (untreated, 1 mM and 5 mM 4-PBA every 2 days). The 4-PBA-treated cells had a clearly reduced growth potential compared to untreated keratinocytes, as visualized by the colony size. Colony size decreased with increasing 4-PBA dosage. B Cells were stained with annexin V / DAPI and then visualized with flow cytometry to detect apoptotic cells. The experiment was done in triplicates with 2 different cell lines for each group. Bar graphs of all experiments show clearly increased numbers of apoptotic cells (especially late apoptotic) in the EBS keratinocyte cell lines compared to NHK; irrespective of the underlying keratin mutations (differences in NHK versus NHK+ [+ standing for cells treated with 1 mM 4-PBA]: necrotic cells P = .1061, late apoptotic cells P = .0955, early apoptotic cells P = .1946 and viable cells P = .0817; differences in KRT14mut versus KRT14mut+: necrotic cells P = .7449, late apoptotic cells * P = .0249, early apoptotic cells P = .9868 and viable cells P = .0979; differences in KRT5mut versus KRT5mut+: necrotic cells P = .5548, late apoptotic cells ** P = .0097, early apoptotic cells P = .1967 and viable cells * P = .0211). Thus, 4-PBA treatment had different effects in NHK and EBS cells. In NHK it induced apoptosis, whereas in the EBS cells it reversed apoptosis. Values represent mean ± S.E.M., unpaired t -test was used. C Total IL1β abundance was evaluated by dot blots in untreated NHK and EBS and 4-PBA treated cells. For the experiment two NHK, two KRT14mut and two KRT5mut cells were used, before and after treatment with 1 mM 4-PBA were used. The experiment was performed in triplicates. The data of the KRT14mut and KRT5mut cells were pulled together for the statistical analysis, since they were similar. Shown above is a representative picture of one NHK and one KRT5mut cell line, before and after 4-PBA treatment. IL1β expression was higher in EBS cells compared to NHK prior to 1 mM 4-PBA treatment, whereas after treatment, IL1β expression was lower in EBS cells compared to NHK cells. Values represent mean ± S.E.M., unpaired t-test was used; for NHK versus EBS ** P = .0096, for EBS+ 1 mM 4-PBA * P = .0257.

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: Effects of 1 mM 4-PBA on cell apoptosis and IL1β expression. A. Colony-forming efficiency assay of 4 different primary NHKs, treated with different dosages of 4-PBA for 1 week is shown (untreated, 1 mM and 5 mM 4-PBA every 2 days). The 4-PBA-treated cells had a clearly reduced growth potential compared to untreated keratinocytes, as visualized by the colony size. Colony size decreased with increasing 4-PBA dosage. B Cells were stained with annexin V / DAPI and then visualized with flow cytometry to detect apoptotic cells. The experiment was done in triplicates with 2 different cell lines for each group. Bar graphs of all experiments show clearly increased numbers of apoptotic cells (especially late apoptotic) in the EBS keratinocyte cell lines compared to NHK; irrespective of the underlying keratin mutations (differences in NHK versus NHK+ [+ standing for cells treated with 1 mM 4-PBA]: necrotic cells P = .1061, late apoptotic cells P = .0955, early apoptotic cells P = .1946 and viable cells P = .0817; differences in KRT14mut versus KRT14mut+: necrotic cells P = .7449, late apoptotic cells * P = .0249, early apoptotic cells P = .9868 and viable cells P = .0979; differences in KRT5mut versus KRT5mut+: necrotic cells P = .5548, late apoptotic cells ** P = .0097, early apoptotic cells P = .1967 and viable cells * P = .0211). Thus, 4-PBA treatment had different effects in NHK and EBS cells. In NHK it induced apoptosis, whereas in the EBS cells it reversed apoptosis. Values represent mean ± S.E.M., unpaired t -test was used. C Total IL1β abundance was evaluated by dot blots in untreated NHK and EBS and 4-PBA treated cells. For the experiment two NHK, two KRT14mut and two KRT5mut cells were used, before and after treatment with 1 mM 4-PBA were used. The experiment was performed in triplicates. The data of the KRT14mut and KRT5mut cells were pulled together for the statistical analysis, since they were similar. Shown above is a representative picture of one NHK and one KRT5mut cell line, before and after 4-PBA treatment. IL1β expression was higher in EBS cells compared to NHK prior to 1 mM 4-PBA treatment, whereas after treatment, IL1β expression was lower in EBS cells compared to NHK cells. Values represent mean ± S.E.M., unpaired t-test was used; for NHK versus EBS ** P = .0096, for EBS+ 1 mM 4-PBA * P = .0257.

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry

    Abundance of significantly affected molecules associated with adhesion and activation of the NF-κB pathway, as found per proteomics, reduced after 1 mM 4-PBA treatment. Quantitative real-time PCR (qPCR) analysis of RNA isolated from keratinocyte cell lines before and after 1 mM 4-PBA 24 h treatment for molecules associated with cell adhesion and activation of the NF-κB pathway. Expression of PPFIA4 , HERC3 , LRP1 and AREG normalized to the expression of GAPDH is shown as the percentage of expression in untreated cells. 4-PBA treatment upregulated the expression of all four genes. Values represent mean ± S.E.M., paired t-test was used [ PPFIA4 : for KRT14mut P = .7074, for KRT5mut * P = .0065; HERC3: for KRT14mut P = .0719, for KRT5mut P = .0657; LRP1: for KRT14mut P = .1811, for KRT5mut P = .1165; AREG: for KRT14mut P = .1007, for KRT5mut P = .3533].

    Journal: EBioMedicine

    Article Title: Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders

    doi: 10.1016/j.ebiom.2019.04.062

    Figure Lengend Snippet: Abundance of significantly affected molecules associated with adhesion and activation of the NF-κB pathway, as found per proteomics, reduced after 1 mM 4-PBA treatment. Quantitative real-time PCR (qPCR) analysis of RNA isolated from keratinocyte cell lines before and after 1 mM 4-PBA 24 h treatment for molecules associated with cell adhesion and activation of the NF-κB pathway. Expression of PPFIA4 , HERC3 , LRP1 and AREG normalized to the expression of GAPDH is shown as the percentage of expression in untreated cells. 4-PBA treatment upregulated the expression of all four genes. Values represent mean ± S.E.M., paired t-test was used [ PPFIA4 : for KRT14mut P = .7074, for KRT5mut * P = .0065; HERC3: for KRT14mut P = .0719, for KRT5mut P = .0657; LRP1: for KRT14mut P = .1811, for KRT5mut P = .1165; AREG: for KRT14mut P = .1007, for KRT5mut P = .3533].

    Article Snippet: Primary keratinocytes from patients' and control skin were cultured in serum-free keratinocyte medium supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor (Invitrogen, Darmstadt, Germany) using standard methods (Kiritsi et al., 2011).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Isolation, Expressing

    Surface expression of integrins in primary keratinocytes isolated from wild-type (lanes 1–3 ) or α3-null (lanes 4–6 ), neonatal mice. Detergent lysates from 125 I surface-labeled cells were immunoprecipitated with antisera against the cytoplasmic domains of the β1, α3, or α6 integrin subunits, as indicated at the top of each lane. Molecular weight markers are shown to the right of the autoradiograph. Migratory positions of certain integrin subunits are indicated to the left, including proteolytic fragments of β4, a , b , and c , described previously by Hemler et al. (1989) . The β1-associated band in α3-null cells that comigrates with α3 (lane 4 ) represents other integrin α subunits that dimerize with β1 in keratinocytes, since α3 was not detected in α3-null cells (lane 5 ). ?, an unidentified band that may represent a proteolytic fragment of β4 (see text).

    Journal: The Journal of Cell Biology

    Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

    doi:

    Figure Lengend Snippet: Surface expression of integrins in primary keratinocytes isolated from wild-type (lanes 1–3 ) or α3-null (lanes 4–6 ), neonatal mice. Detergent lysates from 125 I surface-labeled cells were immunoprecipitated with antisera against the cytoplasmic domains of the β1, α3, or α6 integrin subunits, as indicated at the top of each lane. Molecular weight markers are shown to the right of the autoradiograph. Migratory positions of certain integrin subunits are indicated to the left, including proteolytic fragments of β4, a , b , and c , described previously by Hemler et al. (1989) . The β1-associated band in α3-null cells that comigrates with α3 (lane 4 ) represents other integrin α subunits that dimerize with β1 in keratinocytes, since α3 was not detected in α3-null cells (lane 5 ). ?, an unidentified band that may represent a proteolytic fragment of β4 (see text).

    Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.

    Techniques: Expressing, Isolation, Mouse Assay, Labeling, Immunoprecipitation, Molecular Weight, Autoradiography

    Distributions of α6β4 and laminin-5 in the developing skin of normal and α3-null embryos. Frozen sections from mouse embryonic skin at days E15.5 ( A–F ) or E17.5 ( G–L ) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit ( A , C , and G ) or GoH3 monoclonal antibody against the α6 integrin subunit ( E , I , and K ), and anti–laminin-5 serum ( B , D , H , and F , J , L , respectively). Control sections were from wild-type embryos ( A–D ) or heterozygous embryos ( G and H ). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more stratified regions ( C and D ), but not in less stratified regions ( A and B ); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3-null embryos at E15.5 ( E and F ) and E17.5 ( I and J ), arrowheads point to areas of laminin-5 staining in areas of disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. ( K and L ) Higher magnification of α3-null skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive basement membrane, marked by arrowheads. e , epidermis; d , dermis. Bars: (shown in J for A–J ) 50 μm; and (in L for K and L ) 50 μm.

    Journal: The Journal of Cell Biology

    Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

    doi:

    Figure Lengend Snippet: Distributions of α6β4 and laminin-5 in the developing skin of normal and α3-null embryos. Frozen sections from mouse embryonic skin at days E15.5 ( A–F ) or E17.5 ( G–L ) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit ( A , C , and G ) or GoH3 monoclonal antibody against the α6 integrin subunit ( E , I , and K ), and anti–laminin-5 serum ( B , D , H , and F , J , L , respectively). Control sections were from wild-type embryos ( A–D ) or heterozygous embryos ( G and H ). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more stratified regions ( C and D ), but not in less stratified regions ( A and B ); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3-null embryos at E15.5 ( E and F ) and E17.5 ( I and J ), arrowheads point to areas of laminin-5 staining in areas of disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. ( K and L ) Higher magnification of α3-null skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive basement membrane, marked by arrowheads. e , epidermis; d , dermis. Bars: (shown in J for A–J ) 50 μm; and (in L for K and L ) 50 μm.

    Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.

    Techniques: Staining, Immunofluorescence

    α3β1 is required for postadhesion spreading of mouse keratinocytes on laminin-5. ( A and B ) Primary keratinocytes from neonatal mice heterozygous ( A ) or homozygous ( B ) for the α3null mutation were subcultured on HEK-secreted, laminin-5–rich ECM (see Materials and Methods) and then photographed after 1.5 h. ( C–H ) Primary keratinocytes from wild-type ( C , E , and G ) or α3-null ( D , F , and H ) neonatal mice were subcultured on purified laminin-5 for 1 h and then fixed in 4% paraformaldehyde and stained with Giemsa. ( E and F ) Fibronectin was included with laminin-5 in the substrate. ( G and H ) Cells were treated with the monoclonal antibody GoH3, which blocks adhesion by α6 integrins.

    Journal: The Journal of Cell Biology

    Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

    doi:

    Figure Lengend Snippet: α3β1 is required for postadhesion spreading of mouse keratinocytes on laminin-5. ( A and B ) Primary keratinocytes from neonatal mice heterozygous ( A ) or homozygous ( B ) for the α3null mutation were subcultured on HEK-secreted, laminin-5–rich ECM (see Materials and Methods) and then photographed after 1.5 h. ( C–H ) Primary keratinocytes from wild-type ( C , E , and G ) or α3-null ( D , F , and H ) neonatal mice were subcultured on purified laminin-5 for 1 h and then fixed in 4% paraformaldehyde and stained with Giemsa. ( E and F ) Fibronectin was included with laminin-5 in the substrate. ( G and H ) Cells were treated with the monoclonal antibody GoH3, which blocks adhesion by α6 integrins.

    Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.

    Techniques: Mouse Assay, Mutagenesis, Purification, Staining

    α3-null mice form skin blisters. ( A and B ) Frozen skin sections from wildtype ( A ) or α3-null ( B ) mice were stained with hematoxylin and eosin and the epidermal-dermal junctions were compared. Arrowheads point to basal keratinocytes of the epidermis. ( C and D ) A frozen skin section from an α3null mouse showing a blister viewed by phase contrast ( C ) or stained by immunofluorescence with an antiserum against laminin-5 ( D ). Arrowheads and arrows point to areas of laminin-5 staining at the dermal and epidermal sides of the blister, respectively. e , epidermis; d , dermis. Bars, 50 μm.

    Journal: The Journal of Cell Biology

    Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

    doi:

    Figure Lengend Snippet: α3-null mice form skin blisters. ( A and B ) Frozen skin sections from wildtype ( A ) or α3-null ( B ) mice were stained with hematoxylin and eosin and the epidermal-dermal junctions were compared. Arrowheads point to basal keratinocytes of the epidermis. ( C and D ) A frozen skin section from an α3null mouse showing a blister viewed by phase contrast ( C ) or stained by immunofluorescence with an antiserum against laminin-5 ( D ). Arrowheads and arrows point to areas of laminin-5 staining at the dermal and epidermal sides of the blister, respectively. e , epidermis; d , dermis. Bars, 50 μm.

    Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.

    Techniques: Mouse Assay, Staining, Immunofluorescence

    The basement membrane in α3-null skin is disorganized. ( A–D ) Frozen skin sections from wild-type ( A and C ) or α3-null ( B and D ) mice were viewed by phase contrast ( A and B ) or stained by immunofluorescence with an antiserum against laminin-5 ( C and D ). Arrowheads point to areas of laminin-5 staining at the basement membrane in the wild-type skin ( A and C ) or at regions of disorganized basement membrane in α3-null skin ( B and D ). ( E and F ) Electron micrographs comparing ultrastructure of the basement membrane zone in wild-type ( E ) and α3-null ( F ) skin. ( G and H ) Schematic illustration of relevant structures seen in E and F. BK , basal keratinocyte; LL , lamina lucida; LD , lamina densa; arrowheads point to hemidesmosomes along the basal aspect of the plasma membranes in the basal keratinocytes. Bars: ( D ) 50 μm; ( F ) 200 nm.

    Journal: The Journal of Cell Biology

    Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

    doi:

    Figure Lengend Snippet: The basement membrane in α3-null skin is disorganized. ( A–D ) Frozen skin sections from wild-type ( A and C ) or α3-null ( B and D ) mice were viewed by phase contrast ( A and B ) or stained by immunofluorescence with an antiserum against laminin-5 ( C and D ). Arrowheads point to areas of laminin-5 staining at the basement membrane in the wild-type skin ( A and C ) or at regions of disorganized basement membrane in α3-null skin ( B and D ). ( E and F ) Electron micrographs comparing ultrastructure of the basement membrane zone in wild-type ( E ) and α3-null ( F ) skin. ( G and H ) Schematic illustration of relevant structures seen in E and F. BK , basal keratinocyte; LL , lamina lucida; LD , lamina densa; arrowheads point to hemidesmosomes along the basal aspect of the plasma membranes in the basal keratinocytes. Bars: ( D ) 50 μm; ( F ) 200 nm.

    Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.

    Techniques: Mouse Assay, Staining, Immunofluorescence

    α3β1 integrin is absent from the skin of α3-null mice. Frozen skin sections were prepared from the limbs of wild-type ( A–D ) or α3-null ( E–H ) mice and stained by immunofluorescence with an antiserum against the cytoplasmic domain of α3 ( B and F ), or with the preimmune serum ( D and H ). The corresponding phase contrast ( A , C , E , and G ) is shown to the left of each immunofluorescence panel. e , epidermis; d , dermis. Arrowheads point to basal keratinocytes of the epidermis. Bar, 50 μm.

    Journal: The Journal of Cell Biology

    Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

    doi:

    Figure Lengend Snippet: α3β1 integrin is absent from the skin of α3-null mice. Frozen skin sections were prepared from the limbs of wild-type ( A–D ) or α3-null ( E–H ) mice and stained by immunofluorescence with an antiserum against the cytoplasmic domain of α3 ( B and F ), or with the preimmune serum ( D and H ). The corresponding phase contrast ( A , C , E , and G ) is shown to the left of each immunofluorescence panel. e , epidermis; d , dermis. Arrowheads point to basal keratinocytes of the epidermis. Bar, 50 μm.

    Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.

    Techniques: Mouse Assay, Staining, Immunofluorescence

    The RQ and percentage inhibition of IL-8, CXCL1, and CXCL6 mRNA expression after incubation of NHEK with BCH or pongamia oil for 24 hours in a rosacea environment. IKK inhibitor was used as a positive control. Note: Data shown are the mean of three independent experiments. Abbreviations: RQ, relative quantity; IKK, I kappa B kinase; BCH, 4-t-butylcyclohexanol; NHEK, normal human epidermal keratinocyte.

    Journal: Clinical, Cosmetic and Investigational Dermatology

    Article Title: Effects of dextran sulfate, 4-t-butylcyclohexanol, pongamia oil and hesperidin methyl chalcone on inflammatory and vascular responses implicated in rosacea

    doi: 10.2147/CCID.S168621

    Figure Lengend Snippet: The RQ and percentage inhibition of IL-8, CXCL1, and CXCL6 mRNA expression after incubation of NHEK with BCH or pongamia oil for 24 hours in a rosacea environment. IKK inhibitor was used as a positive control. Note: Data shown are the mean of three independent experiments. Abbreviations: RQ, relative quantity; IKK, I kappa B kinase; BCH, 4-t-butylcyclohexanol; NHEK, normal human epidermal keratinocyte.

    Article Snippet: NHEKs were grown in Keratinocyte Serum-Free Growth Medium (Gibco® , Thermo Fisher Scientific, Waltham, MA, USA) supplemented with bovine pituitary extract and epidermal growth factor (Gibco).

    Techniques: Inhibition, Expressing, Incubation, Positive Control

    Mean (pg/mL) and percentage inhibition of VEGF expression after incubation of keratinocytes with dextran sulfate for 24 hours in a rosacea environment. Note: Data shown are the mean of three independent experiments. ** P

    Journal: Clinical, Cosmetic and Investigational Dermatology

    Article Title: Effects of dextran sulfate, 4-t-butylcyclohexanol, pongamia oil and hesperidin methyl chalcone on inflammatory and vascular responses implicated in rosacea

    doi: 10.2147/CCID.S168621

    Figure Lengend Snippet: Mean (pg/mL) and percentage inhibition of VEGF expression after incubation of keratinocytes with dextran sulfate for 24 hours in a rosacea environment. Note: Data shown are the mean of three independent experiments. ** P

    Article Snippet: NHEKs were grown in Keratinocyte Serum-Free Growth Medium (Gibco® , Thermo Fisher Scientific, Waltham, MA, USA) supplemented with bovine pituitary extract and epidermal growth factor (Gibco).

    Techniques: Inhibition, Expressing, Incubation

    Serum and Ca ++ affect growth and TGFβ-induced hypermotility differently a,b. Effects of [Ca ++ ] and serum on keratinocyte and prostate epithelial cell growth. Panel a: G5Ep and HPrE-1 cells plated at low density (~250 cells/cm 2 ) in Ksfm supplemented with 0.1, 0.4, or 1.0 mM Ca ++ or with 0.4 mM Ca ++ and 1% calf serum. Note that both cell types grew as well or better in higher [Ca ++ ] but were strongly inhibited by addition of 1% serum to the medium. Panel b: typical fields of HPrE-1 cells in the various conditions. Note inability to form closely packed colonies in low and medium [Ca ++ ] and abnormal elongation associated with growth inhibition in presence of serum. c. Increased sensitivity to TGFβ growth inhibition of keratinocyte and prostate epithelial cells when plated on serum-precoated dishes. Cells were plated in Ksfm in wells that were not pretreated or that were precoated with 10% serum. Note little change in growth rate −TGFβ and a significant dose-response shift toward increased TGFβ-induced growth inhibition for both cell types on serum-precoated dishes. d. A substratum-bound component of serum blocks TGFβ-induced hypermotility. G5Ep and HPrE-1 cells were plated + or −TGFβ in Ksfm with 0.2 mM Ca ++ in untreated or serum-precoated wells. Note lack of hypermotility response to TGFβ of cells in serum-precoated wells. e. TGFβ-induced hypermotility is inversely related to [Ca ++ ] of the medium. G5Ep cells were plated + or −TGFβ or on Lam332′-precoated surfaces in Ksfm with [Ca ++ ] = 0.1 mM, 0.4 mM, or 1.0 mM. Note that hypermotility induced by TGFβ was greatly reduced, and that by Lam332′ slightly attenuated, by increasing [Ca ++ ].

    Journal: In vitro cellular & developmental biology. Animal

    Article Title: Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

    doi: 10.1007/s11626-010-9353-8

    Figure Lengend Snippet: Serum and Ca ++ affect growth and TGFβ-induced hypermotility differently a,b. Effects of [Ca ++ ] and serum on keratinocyte and prostate epithelial cell growth. Panel a: G5Ep and HPrE-1 cells plated at low density (~250 cells/cm 2 ) in Ksfm supplemented with 0.1, 0.4, or 1.0 mM Ca ++ or with 0.4 mM Ca ++ and 1% calf serum. Note that both cell types grew as well or better in higher [Ca ++ ] but were strongly inhibited by addition of 1% serum to the medium. Panel b: typical fields of HPrE-1 cells in the various conditions. Note inability to form closely packed colonies in low and medium [Ca ++ ] and abnormal elongation associated with growth inhibition in presence of serum. c. Increased sensitivity to TGFβ growth inhibition of keratinocyte and prostate epithelial cells when plated on serum-precoated dishes. Cells were plated in Ksfm in wells that were not pretreated or that were precoated with 10% serum. Note little change in growth rate −TGFβ and a significant dose-response shift toward increased TGFβ-induced growth inhibition for both cell types on serum-precoated dishes. d. A substratum-bound component of serum blocks TGFβ-induced hypermotility. G5Ep and HPrE-1 cells were plated + or −TGFβ in Ksfm with 0.2 mM Ca ++ in untreated or serum-precoated wells. Note lack of hypermotility response to TGFβ of cells in serum-precoated wells. e. TGFβ-induced hypermotility is inversely related to [Ca ++ ] of the medium. G5Ep cells were plated + or −TGFβ or on Lam332′-precoated surfaces in Ksfm with [Ca ++ ] = 0.1 mM, 0.4 mM, or 1.0 mM. Note that hypermotility induced by TGFβ was greatly reduced, and that by Lam332′ slightly attenuated, by increasing [Ca ++ ].

    Article Snippet: The normal human newborn foreskin epidermal keratinocyte primary line G5Ep, which we initiated, was cultured in keratinocyte serum-free medium (Ksfm) (GIBCO/Invitrogen, Carlsbad, CA), supplemented as described previously [ ; ] with 25 μg/ml bovine pituitary extract (BPE) (GIBCO), 0.2 ng/ml epidermal growth factor (EGF) (R & D Systems, Minneapolis, MN), 0.3 mM CaCl2 (to bring the [Ca++ ] to 0.4 mM), and penicillin-streptomycin (GIBCO).

    Techniques: Inhibition

    TGFβ-induced responses and TGFβ receptor-dependent signaling in normal primary and immortalized prostate epithelial cells a. TGFβ-induced growth inhibition. Note that HPrE-1/bmi1/TERT cells (HPrE-1/b/T) remained sensitive to TGFβ growth inhibition, although their dose-response was shifted about 3-fold toward reduced sensitivity compared with HPrE-1 cells. In contrast, PC3 cells were completely resistant to TGFβ. b. TGFβ and Lam332′-induced hypermotility. Note that HPrE-1/bmi1/TERT cells remained sensitive to TGFβ- and Lam332′-induced hypermotility. c. Smad2 phosphorylation induced by TGFβ in normal primary keratinocytes and prostate epithelial cells and in experimentally immortalized prostate epithelial cells. Preconfluent cultures of G5Ep, HPrE-1, and HPrE-1/bmi1/TERT growing in Ksfm were untreated or treated with 0.1 ng/ml TGFβ for the final 30 min before lysis and analysis by Western blotting. Note very low or undetectable constitutive, and TGFβ-activated, smad2 phosphorylation in all three cell lines. d. Defects in TGFβ-induced smad signaling and p63 and Lam332 expression by prostate carcinoma cell lines. Preconfluent cultures were untreated or treated with 0.1 ng/ml TGFβ for the final 24 hr before lysis and analysis by Western blotting. Note absence of p63 expression by the prostate carcinoma lines. The Laminin γ2 blot was overexposed for HPrE1 to permit detection of very low levels of γ2 expression and its induction by TGFβ in PC3, consistent with smad2 phosphorylation stimulated by TGFβ in this line. In contrast, MDAPCa-2b and LNCaP did not phosphorylate smad2 in response to TGFβ and did not synthesize detectable γ2.

    Journal: In vitro cellular & developmental biology. Animal

    Article Title: Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

    doi: 10.1007/s11626-010-9353-8

    Figure Lengend Snippet: TGFβ-induced responses and TGFβ receptor-dependent signaling in normal primary and immortalized prostate epithelial cells a. TGFβ-induced growth inhibition. Note that HPrE-1/bmi1/TERT cells (HPrE-1/b/T) remained sensitive to TGFβ growth inhibition, although their dose-response was shifted about 3-fold toward reduced sensitivity compared with HPrE-1 cells. In contrast, PC3 cells were completely resistant to TGFβ. b. TGFβ and Lam332′-induced hypermotility. Note that HPrE-1/bmi1/TERT cells remained sensitive to TGFβ- and Lam332′-induced hypermotility. c. Smad2 phosphorylation induced by TGFβ in normal primary keratinocytes and prostate epithelial cells and in experimentally immortalized prostate epithelial cells. Preconfluent cultures of G5Ep, HPrE-1, and HPrE-1/bmi1/TERT growing in Ksfm were untreated or treated with 0.1 ng/ml TGFβ for the final 30 min before lysis and analysis by Western blotting. Note very low or undetectable constitutive, and TGFβ-activated, smad2 phosphorylation in all three cell lines. d. Defects in TGFβ-induced smad signaling and p63 and Lam332 expression by prostate carcinoma cell lines. Preconfluent cultures were untreated or treated with 0.1 ng/ml TGFβ for the final 24 hr before lysis and analysis by Western blotting. Note absence of p63 expression by the prostate carcinoma lines. The Laminin γ2 blot was overexposed for HPrE1 to permit detection of very low levels of γ2 expression and its induction by TGFβ in PC3, consistent with smad2 phosphorylation stimulated by TGFβ in this line. In contrast, MDAPCa-2b and LNCaP did not phosphorylate smad2 in response to TGFβ and did not synthesize detectable γ2.

    Article Snippet: The normal human newborn foreskin epidermal keratinocyte primary line G5Ep, which we initiated, was cultured in keratinocyte serum-free medium (Ksfm) (GIBCO/Invitrogen, Carlsbad, CA), supplemented as described previously [ ; ] with 25 μg/ml bovine pituitary extract (BPE) (GIBCO), 0.2 ng/ml epidermal growth factor (EGF) (R & D Systems, Minneapolis, MN), 0.3 mM CaCl2 (to bring the [Ca++ ] to 0.4 mM), and penicillin-streptomycin (GIBCO).

    Techniques: Inhibition, Lysis, Western Blot, Expressing

    Colony morphology and marker expression by cultured human keratinocytes and prostate epithelial cells a. Phase contrast images of typical colonies formed by G5Ep keratinocytes and HPrE-1 prostate epithelial cells one week after plating in Ksfm or PrEGM medium. Note the more stable cell-cell association by both cell types in the 0.4 mM Ca ++ Ksfm than in the 0.1 mM Ca ++ PrEGM medium and the less scattered morphology of G5Ep compared to HPrE-1 cells in both media. Bar in upper left panel: 200 microns. b. G5Ep and HPrE-1 cultures immunostained for p63, β4 integrin, and the γ2 chain of Lam332. Note expression of these proteins by all cells of both types and that Lam332 is detectable in the cytoplasm and deposited by cells onto the dish surface. Bar in upper left panel: 200 microns.

    Journal: In vitro cellular & developmental biology. Animal

    Article Title: Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

    doi: 10.1007/s11626-010-9353-8

    Figure Lengend Snippet: Colony morphology and marker expression by cultured human keratinocytes and prostate epithelial cells a. Phase contrast images of typical colonies formed by G5Ep keratinocytes and HPrE-1 prostate epithelial cells one week after plating in Ksfm or PrEGM medium. Note the more stable cell-cell association by both cell types in the 0.4 mM Ca ++ Ksfm than in the 0.1 mM Ca ++ PrEGM medium and the less scattered morphology of G5Ep compared to HPrE-1 cells in both media. Bar in upper left panel: 200 microns. b. G5Ep and HPrE-1 cultures immunostained for p63, β4 integrin, and the γ2 chain of Lam332. Note expression of these proteins by all cells of both types and that Lam332 is detectable in the cytoplasm and deposited by cells onto the dish surface. Bar in upper left panel: 200 microns.

    Article Snippet: The normal human newborn foreskin epidermal keratinocyte primary line G5Ep, which we initiated, was cultured in keratinocyte serum-free medium (Ksfm) (GIBCO/Invitrogen, Carlsbad, CA), supplemented as described previously [ ; ] with 25 μg/ml bovine pituitary extract (BPE) (GIBCO), 0.2 ng/ml epidermal growth factor (EGF) (R & D Systems, Minneapolis, MN), 0.3 mM CaCl2 (to bring the [Ca++ ] to 0.4 mM), and penicillin-streptomycin (GIBCO).

    Techniques: Marker, Expressing, Cell Culture, Stable Transfection

    The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human keratinocytes

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human keratinocytes

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Dominant Negative Mutation, Blocking Assay, Activity Assay, Inhibition, Cell Culture

    PI3K inhibition blocks the stimulation of PLC-γ1 activity by calcium. Cultured human keratinocytes were preincubated with 10 μM LY294002 (+) or vehicle DMSO (–) for 1 h. Cells were then stimulated with 1.2 mM calcium for 1 h, and

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: PI3K inhibition blocks the stimulation of PLC-γ1 activity by calcium. Cultured human keratinocytes were preincubated with 10 μM LY294002 (+) or vehicle DMSO (–) for 1 h. Cells were then stimulated with 1.2 mM calcium for 1 h, and

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Inhibition, Planar Chromatography, Activity Assay, Cell Culture

    The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn block calcium-induced involucrin and transglutaminase expression. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2)

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn block calcium-induced involucrin and transglutaminase expression. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2)

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Dominant Negative Mutation, Blocking Assay, Expressing, Cell Culture

    Calcium stimulates PI3K-p85α phosphorylation, PI3K activity, and Akt phosphorylation. (A). Cultured human keratinocytes were treated with 1.2 mM calcium for the indicated times. The protein levels of PI3K-p85α and its phosphorylated form

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium stimulates PI3K-p85α phosphorylation, PI3K activity, and Akt phosphorylation. (A). Cultured human keratinocytes were treated with 1.2 mM calcium for the indicated times. The protein levels of PI3K-p85α and its phosphorylated form

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Activity Assay, Cell Culture

    Calcium does not stimulate the phosphorylation of PLC-γ1. Cultured human keratinocytes were treated with 1.2 mM calcium or 50 ng/ml EGF. The cells were harvested at the time points indicated. The protein levels of PLC-γ1 and its phosphorylated

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium does not stimulate the phosphorylation of PLC-γ1. Cultured human keratinocytes were treated with 1.2 mM calcium or 50 ng/ml EGF. The cells were harvested at the time points indicated. The protein levels of PLC-γ1 and its phosphorylated

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Cell Culture

    Calcium stimulates PLC-γ1 activity. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated after the addition of calcium. The activity of PLC-γ1 was assayed as described in

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium stimulates PLC-γ1 activity. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated after the addition of calcium. The activity of PLC-γ1 was assayed as described in

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Activity Assay, Cell Culture

    The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn reduce calcium-stimulated PLC-γ1 activity. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2) or its inactive

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn reduce calcium-stimulated PLC-γ1 activity. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2) or its inactive

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Dominant Negative Mutation, Planar Chromatography, Activity Assay, Cell Culture

    Calcium stimulates src and fyn activities. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated. The activities of src (A) and fyn (B) were assayed as described in Materials and Methods and

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium stimulates src and fyn activities. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated. The activities of src (A) and fyn (B) were assayed as described in Materials and Methods and

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Cell Culture

    Proposed model for the signaling pathway of PLC-γ1 activation mediating calcium-induced human keratinocyte differentiation. Calcium (Ca 2+ ) stimulates src and fyn , which activate PI3K via

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Proposed model for the signaling pathway of PLC-γ1 activation mediating calcium-induced human keratinocyte differentiation. Calcium (Ca 2+ ) stimulates src and fyn , which activate PI3K via

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Activation Assay

    PI3K inhibition blocks the induction of involucrin and transglutaminase expression by calcium. Cultured human keratinocytes were preincubated with 10 mM LY294002 (+) or vehicle DMSO (–) for 1 h and then treated with 1.2 mM calcium for 24 h. The

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: PI3K inhibition blocks the induction of involucrin and transglutaminase expression by calcium. Cultured human keratinocytes were preincubated with 10 mM LY294002 (+) or vehicle DMSO (–) for 1 h and then treated with 1.2 mM calcium for 24 h. The

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Inhibition, Expressing, Cell Culture

    PIP 3 stimulates PLC-γ1 activity, and the binding of PIP 3 to PLC-γ1 is induced by calcium. (A). Human keratinocytes were cultured in KGM with 0.03 mM calcium. The total cell lysate was isolated with the cell lysis buffer containing 50 mM

    Journal: Molecular Biology of the Cell

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: PIP 3 stimulates PLC-γ1 activity, and the binding of PIP 3 to PLC-γ1 is induced by calcium. (A). Human keratinocytes were cultured in KGM with 0.03 mM calcium. The total cell lysate was isolated with the cell lysis buffer containing 50 mM

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Activity Assay, Binding Assay, Cell Culture, Isolation, Lysis

    An EMT-like phenotype was observed in the GnT-V Tg mouse keratinocytes. The EMT-associated transcriptional factors snail, twist, E-cadherin, and, N-cadherin were evaluated by quantitative RT-PCR, and target gene expression was normalized to GAPDH. Results

    Journal: The Journal of Biological Chemistry

    Article Title: Enhanced Epithelial-Mesenchymal Transition-like Phenotype in N-Acetylglucosaminyltransferase V Transgenic Mouse Skin Promotes Wound Healing *

    doi: 10.1074/jbc.M111.220376

    Figure Lengend Snippet: An EMT-like phenotype was observed in the GnT-V Tg mouse keratinocytes. The EMT-associated transcriptional factors snail, twist, E-cadherin, and, N-cadherin were evaluated by quantitative RT-PCR, and target gene expression was normalized to GAPDH. Results

    Article Snippet: Cells were incubated in human keratinocyte serum-free medium (DS Pharma Biomedical, Osaka, Japan) for 6 to 12 h at 37 °C in 5% CO2 to allow the cells to adhere to culture dishes precoated with type 1 collagen (Asahi Techno Glass, Funabashi, Japan).

    Techniques: Quantitative RT-PCR, Expressing

    Increased β1,6 GlcNAc branching on the EGF-R in GnT-V Tg mouse keratinocytes up-regulates EGF-R signaling. A , Western blot analysis of EGF-R after L 4 -PHA lectin immunoprecipitation of skin specimens from wild-type and GnT-V Tg mice ( n = 2/each

    Journal: The Journal of Biological Chemistry

    Article Title: Enhanced Epithelial-Mesenchymal Transition-like Phenotype in N-Acetylglucosaminyltransferase V Transgenic Mouse Skin Promotes Wound Healing *

    doi: 10.1074/jbc.M111.220376

    Figure Lengend Snippet: Increased β1,6 GlcNAc branching on the EGF-R in GnT-V Tg mouse keratinocytes up-regulates EGF-R signaling. A , Western blot analysis of EGF-R after L 4 -PHA lectin immunoprecipitation of skin specimens from wild-type and GnT-V Tg mice ( n = 2/each

    Article Snippet: Cells were incubated in human keratinocyte serum-free medium (DS Pharma Biomedical, Osaka, Japan) for 6 to 12 h at 37 °C in 5% CO2 to allow the cells to adhere to culture dishes precoated with type 1 collagen (Asahi Techno Glass, Funabashi, Japan).

    Techniques: Western Blot, Immunoprecipitation, Mouse Assay

    Enhanced migration of GnT-V Tg mouse keratinocytes. Scrape-wounded, confluent keratinocyte monolayers of wild-type and Tg mice were incubated. A and B , phase-contrast microscopy images of scratch wounds at baseline and at 24 h. Results are representative

    Journal: The Journal of Biological Chemistry

    Article Title: Enhanced Epithelial-Mesenchymal Transition-like Phenotype in N-Acetylglucosaminyltransferase V Transgenic Mouse Skin Promotes Wound Healing *

    doi: 10.1074/jbc.M111.220376

    Figure Lengend Snippet: Enhanced migration of GnT-V Tg mouse keratinocytes. Scrape-wounded, confluent keratinocyte monolayers of wild-type and Tg mice were incubated. A and B , phase-contrast microscopy images of scratch wounds at baseline and at 24 h. Results are representative

    Article Snippet: Cells were incubated in human keratinocyte serum-free medium (DS Pharma Biomedical, Osaka, Japan) for 6 to 12 h at 37 °C in 5% CO2 to allow the cells to adhere to culture dishes precoated with type 1 collagen (Asahi Techno Glass, Funabashi, Japan).

    Techniques: Migration, Mouse Assay, Incubation, Microscopy

    An EMT-like phenotype was observed in GnT-V Tg mouse keratinocytes. A, B, C, and D , primary cultured keratinocytes derived from wild-type and GnT-V Tg mice were stained with anti-E-cadherin ( A , green), anti-N-cadherin ( B , green ), anti-α-SMA (

    Journal: The Journal of Biological Chemistry

    Article Title: Enhanced Epithelial-Mesenchymal Transition-like Phenotype in N-Acetylglucosaminyltransferase V Transgenic Mouse Skin Promotes Wound Healing *

    doi: 10.1074/jbc.M111.220376

    Figure Lengend Snippet: An EMT-like phenotype was observed in GnT-V Tg mouse keratinocytes. A, B, C, and D , primary cultured keratinocytes derived from wild-type and GnT-V Tg mice were stained with anti-E-cadherin ( A , green), anti-N-cadherin ( B , green ), anti-α-SMA (

    Article Snippet: Cells were incubated in human keratinocyte serum-free medium (DS Pharma Biomedical, Osaka, Japan) for 6 to 12 h at 37 °C in 5% CO2 to allow the cells to adhere to culture dishes precoated with type 1 collagen (Asahi Techno Glass, Funabashi, Japan).

    Techniques: Cell Culture, Derivative Assay, Mouse Assay, Staining