keratinocyte-serum free medium Search Results


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  • 99
    Thermo Fisher keratinocyte sfm medium
    Primary human NPC cells cultured in <t>keratinocyte-SFM</t> medium. (A) Magnification, ×100; (B) magnification, ×400; (C) magnification, ×400 (Giemsa staining). (D and E) Cytokeratin expression in primary human NPC cells. (F) The growth curve of CD133 + and CD133 − cells in primary NPC cells.
    Keratinocyte Sfm Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1619 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1619 article reviews
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    keratinocyte sfm medium - by Bioz Stars, 2020-09
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    98
    Millipore keratinocyte serum free medium
    EM detection of TG1-FLAG and TG1-FLAG(C377A). <t>Keratinocytes</t> were infected with empty virus ( EV ) or virus encoding TG1-FLAG or TG1-FLAG(C377A) and then processed for transmission electron microscopy. The ER and nucleus ( N ) are indicated. The arrows in the enlarged panels indicate clusters of colloidal gold particles localized in the ER. Bars = 500 nm.
    Keratinocyte Serum Free Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 46 article reviews
    Price from $9.99 to $1999.99
    keratinocyte serum free medium - by Bioz Stars, 2020-09
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    95
    Thermo Fisher keratinocyte serum free medium
    KLF4 is critical for IL-36γ transcriptional activity upon EGFR/MEK inhibition. ( A ) KLF4-overexpressing primary keratinocytes were exposed to C . acnes for 24 hours. ( B ) Flag-tagged wild-type (WT) and dominant-negative (DN) KLF4 were overexpressed in response to doxycycline using a Tet-on system for 24 hours, followed by exposure to Pam3CSK4 for another 24 hours. The cell lysates were collected for Western blotting and quantitative PCR (qPCR). Data represent mean ± SEM ( n = 3). ( C ) KLF4 siRNA–treated PHKs were exposed to erlotinib and C . acnes for 6 hours and IL36G levels were analyzed by qPCR. Data represent mean ± SEM ( n = 3). ( D ) <t>Keratinocyte</t> cell lines in which KLF4 was knocked out by CRISPR/Cas9 were exposed to trametinib (2 μg/mL) for 24 hours and total cell lysates were collected for Western blotting with antibodies against KLF4 and β-actin. The cells were exposed to trametinib for 24 hours and isolated RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). All blots were run contemporaneously with the same protein samples. Data are representative of 3 independent experiments. ( E ) Mutations generated by CRISPR/Cas9 in the KLF4 binding site. Red nucleotides are the PAM sequence and blue nucleotides hybridize to the sgRNA. KLF4 binding site–mutant cells were exposed to trametinib and Pam3CSK4 for 24 hours. Total RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). Data were analyzed with 1-way ANOVA followed by Dunnett’s ( B and E ) or Tukey’s multiple-comparisons test ( C ) or with 2-tailed unpaired t test ( D ). ** P
    Keratinocyte Serum Free Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 6805 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratinocyte serum free medium/product/Thermo Fisher
    Average 95 stars, based on 6805 article reviews
    Price from $9.99 to $1999.99
    keratinocyte serum free medium - by Bioz Stars, 2020-09
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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
    https://www.bioz.com/result/keratinocyte serum free medium ksfm/product/Thermo Fisher
    Average 91 stars, based on 685 article reviews
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    99
    Thermo Fisher keratinocyte sfm media
    <t>Keratinocytes</t> and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for
    Keratinocyte Sfm Media, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratinocyte sfm media/product/Thermo Fisher
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    keratinocyte sfm media - by Bioz Stars, 2020-09
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    89
    Thermo Fisher keratinocyte serum free media k sfm
    <t>Keratinocytes</t> and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for
    Keratinocyte Serum Free Media K Sfm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ScienCell serum free keratinocyte medium
    <t>Keratinocytes</t> and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for
    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
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    90
    Thermo Fisher keratinocyte serum free ksf medium
    <t>Keratinocytes</t> and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for
    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
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    95
    Thermo Fisher keratinocyte sfm 1x without calcium chloride
    <t>Keratinocytes</t> and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for
    Keratinocyte Sfm 1x Without Calcium Chloride, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    Image Search Results


    Primary human NPC cells cultured in keratinocyte-SFM medium. (A) Magnification, ×100; (B) magnification, ×400; (C) magnification, ×400 (Giemsa staining). (D and E) Cytokeratin expression in primary human NPC cells. (F) The growth curve of CD133 + and CD133 − cells in primary NPC cells.

    Journal: Oncology Reports

    Article Title: Biological characteristics of CD133+ cells in nasopharyngeal carcinoma

    doi: 10.3892/or.2013.2408

    Figure Lengend Snippet: Primary human NPC cells cultured in keratinocyte-SFM medium. (A) Magnification, ×100; (B) magnification, ×400; (C) magnification, ×400 (Giemsa staining). (D and E) Cytokeratin expression in primary human NPC cells. (F) The growth curve of CD133 + and CD133 − cells in primary NPC cells.

    Article Snippet: At 4 weeks after the transplantation, the xenograft tumours were collected and fixed in 4% paraformaldehyde for H & E staining or were incubated in explant tissue cultures with 3 ml of keratinocyte-SFM medium (Gibco) for cell culture.

    Techniques: Cell Culture, Staining, Expressing

    EM detection of TG1-FLAG and TG1-FLAG(C377A). Keratinocytes were infected with empty virus ( EV ) or virus encoding TG1-FLAG or TG1-FLAG(C377A) and then processed for transmission electron microscopy. The ER and nucleus ( N ) are indicated. The arrows in the enlarged panels indicate clusters of colloidal gold particles localized in the ER. Bars = 500 nm.

    Journal: The Journal of Biological Chemistry

    Article Title: Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis *

    doi: 10.1074/jbc.M110.128645

    Figure Lengend Snippet: EM detection of TG1-FLAG and TG1-FLAG(C377A). Keratinocytes were infected with empty virus ( EV ) or virus encoding TG1-FLAG or TG1-FLAG(C377A) and then processed for transmission electron microscopy. The ER and nucleus ( N ) are indicated. The arrows in the enlarged panels indicate clusters of colloidal gold particles localized in the ER. Bars = 500 nm.

    Article Snippet: For experiments, keratinocytes were incubated with 2.5–10 m.o.i. of tAd5-EV or tAd5-TG1-FLAG in the presence of 5 m.o.i. of Ad5-transactivator in keratinocyte serum-free medium containing 6 μg/ml of Polybrene (Sigma). pcDNA3-TG1-FLAG(R142C) and pcDNA3-TG1-FLAG(V379L) were produced by site-directed mutagenesis to convert R142C and V379L. pcDNA3-TG1-FLAG(C377A) and pΔE1Sp1Btet-TG1-FLAG(C377A) were produced by site-directed mutagenesis to convert cysteine 377 of TG1 to alanine. pΔE1Sp1Btet-TG1-FLAG(C377A) was then packaged with the pJM17 adenovirus backbone to produce the tAd5-TG1-FLAG(C377A) adenovirus.

    Techniques: Infection, Transmission Assay, Electron Microscopy

    Subcellular location of TG1 mutants. A , structure of TG1 showing the anchor, β-sandwich, catalytic, and β-barrel 1 and β-barrel 2 domains and the three residues that comprise the catalytic triad (Cys 377 , His 435 , and Asp 459 ) and the four ( C47 , 48 , 50 , 51 ) of the five cysteines that form the site that is lipid modified to add the membrane anchor. The position of the FLAG epitope tag is indicated. The numbers at the top indicate amino acids residue. The site of the mutation in TG1-FLAG(C377A) is indicated. FLAG-TG1(Δ1–52) is a truncation mutant. B , subcellular distribution of TG1-FLAG(C377A). Keratinocytes on coverslips were transfected with 2 μg of pcDNA3-TG1-FLAG(C377A) and after 48 h fixed and stained with anti-FLAG ( green ). The z -stack of images was generated with the bottom picture being at the coverslip/cell junction. C , cells were transfected with 2 μg of pcDNA3-FLAG-TG1(Δ1–52) and after 48 h fixed and stained with anti-FLAG. Nuclei were visualized using Hoescht stain. The bar = 10 μm in all panels. D , to monitor TG1-FLAG, TG1-FLAG(C377A) and FLAG-TG1(Δ1–52) expression cells were transfected with 10 μg of the indicated plasmid and after 48 h extracts were prepared for anti-FLAG immunoblot. E , cells were transfected with plasmids encoding TG1(C377A)(no tag), TG1-FLAG(R142C), and TG1-FLAG(V379L) and stained at 24 h with anti-TG1 ( left panel ) or anti-FLAG ( middle and right panels ). Nuclei were visualized using Hoescht stain. All images are 1-μm confocal optical sections.

    Journal: The Journal of Biological Chemistry

    Article Title: Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis *

    doi: 10.1074/jbc.M110.128645

    Figure Lengend Snippet: Subcellular location of TG1 mutants. A , structure of TG1 showing the anchor, β-sandwich, catalytic, and β-barrel 1 and β-barrel 2 domains and the three residues that comprise the catalytic triad (Cys 377 , His 435 , and Asp 459 ) and the four ( C47 , 48 , 50 , 51 ) of the five cysteines that form the site that is lipid modified to add the membrane anchor. The position of the FLAG epitope tag is indicated. The numbers at the top indicate amino acids residue. The site of the mutation in TG1-FLAG(C377A) is indicated. FLAG-TG1(Δ1–52) is a truncation mutant. B , subcellular distribution of TG1-FLAG(C377A). Keratinocytes on coverslips were transfected with 2 μg of pcDNA3-TG1-FLAG(C377A) and after 48 h fixed and stained with anti-FLAG ( green ). The z -stack of images was generated with the bottom picture being at the coverslip/cell junction. C , cells were transfected with 2 μg of pcDNA3-FLAG-TG1(Δ1–52) and after 48 h fixed and stained with anti-FLAG. Nuclei were visualized using Hoescht stain. The bar = 10 μm in all panels. D , to monitor TG1-FLAG, TG1-FLAG(C377A) and FLAG-TG1(Δ1–52) expression cells were transfected with 10 μg of the indicated plasmid and after 48 h extracts were prepared for anti-FLAG immunoblot. E , cells were transfected with plasmids encoding TG1(C377A)(no tag), TG1-FLAG(R142C), and TG1-FLAG(V379L) and stained at 24 h with anti-TG1 ( left panel ) or anti-FLAG ( middle and right panels ). Nuclei were visualized using Hoescht stain. All images are 1-μm confocal optical sections.

    Article Snippet: For experiments, keratinocytes were incubated with 2.5–10 m.o.i. of tAd5-EV or tAd5-TG1-FLAG in the presence of 5 m.o.i. of Ad5-transactivator in keratinocyte serum-free medium containing 6 μg/ml of Polybrene (Sigma). pcDNA3-TG1-FLAG(R142C) and pcDNA3-TG1-FLAG(V379L) were produced by site-directed mutagenesis to convert R142C and V379L. pcDNA3-TG1-FLAG(C377A) and pΔE1Sp1Btet-TG1-FLAG(C377A) were produced by site-directed mutagenesis to convert cysteine 377 of TG1 to alanine. pΔE1Sp1Btet-TG1-FLAG(C377A) was then packaged with the pJM17 adenovirus backbone to produce the tAd5-TG1-FLAG(C377A) adenovirus.

    Techniques: Modification, FLAG-tag, Mutagenesis, Transfection, Staining, Generated, Expressing, Plasmid Preparation

    TG1-FLAG(C377A) produces a unique phenotype. A , keratinocytes were infected with 10 m.o.i. of tAd5-EV, tAd5-TG1-FLAG, or tAd5-TG1-FLAG(C377A) and at 72 h fixed and stained for morphology assessment and detection of FLAG epitope. The arrows in the TG1 panel identify blebs. B , expression level of TG1 mutants was monitored by immunoblot of total extracts. C , impact on chromatin. Keratinocytes were infected with adenovirus encoding the indicated protein were fixed at 72 h and stained with Hoechst. The percent of cells displaying each nuclear phenotype was determined by counting cells: EV , 265 cells in ten fields; TG1-FLAG , 223 cells in ten fields; TG1-FLAG ( C377A ), 162 cells in nine fields. Bar = 10 μm. The terms normal , dispersed , and compact are operational descriptions of the chromatin phenotype.

    Journal: The Journal of Biological Chemistry

    Article Title: Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis *

    doi: 10.1074/jbc.M110.128645

    Figure Lengend Snippet: TG1-FLAG(C377A) produces a unique phenotype. A , keratinocytes were infected with 10 m.o.i. of tAd5-EV, tAd5-TG1-FLAG, or tAd5-TG1-FLAG(C377A) and at 72 h fixed and stained for morphology assessment and detection of FLAG epitope. The arrows in the TG1 panel identify blebs. B , expression level of TG1 mutants was monitored by immunoblot of total extracts. C , impact on chromatin. Keratinocytes were infected with adenovirus encoding the indicated protein were fixed at 72 h and stained with Hoechst. The percent of cells displaying each nuclear phenotype was determined by counting cells: EV , 265 cells in ten fields; TG1-FLAG , 223 cells in ten fields; TG1-FLAG ( C377A ), 162 cells in nine fields. Bar = 10 μm. The terms normal , dispersed , and compact are operational descriptions of the chromatin phenotype.

    Article Snippet: For experiments, keratinocytes were incubated with 2.5–10 m.o.i. of tAd5-EV or tAd5-TG1-FLAG in the presence of 5 m.o.i. of Ad5-transactivator in keratinocyte serum-free medium containing 6 μg/ml of Polybrene (Sigma). pcDNA3-TG1-FLAG(R142C) and pcDNA3-TG1-FLAG(V379L) were produced by site-directed mutagenesis to convert R142C and V379L. pcDNA3-TG1-FLAG(C377A) and pΔE1Sp1Btet-TG1-FLAG(C377A) were produced by site-directed mutagenesis to convert cysteine 377 of TG1 to alanine. pΔE1Sp1Btet-TG1-FLAG(C377A) was then packaged with the pJM17 adenovirus backbone to produce the tAd5-TG1-FLAG(C377A) adenovirus.

    Techniques: Infection, Staining, FLAG-tag, Expressing

    KLF4 is critical for IL-36γ transcriptional activity upon EGFR/MEK inhibition. ( A ) KLF4-overexpressing primary keratinocytes were exposed to C . acnes for 24 hours. ( B ) Flag-tagged wild-type (WT) and dominant-negative (DN) KLF4 were overexpressed in response to doxycycline using a Tet-on system for 24 hours, followed by exposure to Pam3CSK4 for another 24 hours. The cell lysates were collected for Western blotting and quantitative PCR (qPCR). Data represent mean ± SEM ( n = 3). ( C ) KLF4 siRNA–treated PHKs were exposed to erlotinib and C . acnes for 6 hours and IL36G levels were analyzed by qPCR. Data represent mean ± SEM ( n = 3). ( D ) Keratinocyte cell lines in which KLF4 was knocked out by CRISPR/Cas9 were exposed to trametinib (2 μg/mL) for 24 hours and total cell lysates were collected for Western blotting with antibodies against KLF4 and β-actin. The cells were exposed to trametinib for 24 hours and isolated RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). All blots were run contemporaneously with the same protein samples. Data are representative of 3 independent experiments. ( E ) Mutations generated by CRISPR/Cas9 in the KLF4 binding site. Red nucleotides are the PAM sequence and blue nucleotides hybridize to the sgRNA. KLF4 binding site–mutant cells were exposed to trametinib and Pam3CSK4 for 24 hours. Total RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). Data were analyzed with 1-way ANOVA followed by Dunnett’s ( B and E ) or Tukey’s multiple-comparisons test ( C ) or with 2-tailed unpaired t test ( D ). ** P

    Journal: The Journal of Clinical Investigation

    Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

    doi: 10.1172/JCI128678

    Figure Lengend Snippet: KLF4 is critical for IL-36γ transcriptional activity upon EGFR/MEK inhibition. ( A ) KLF4-overexpressing primary keratinocytes were exposed to C . acnes for 24 hours. ( B ) Flag-tagged wild-type (WT) and dominant-negative (DN) KLF4 were overexpressed in response to doxycycline using a Tet-on system for 24 hours, followed by exposure to Pam3CSK4 for another 24 hours. The cell lysates were collected for Western blotting and quantitative PCR (qPCR). Data represent mean ± SEM ( n = 3). ( C ) KLF4 siRNA–treated PHKs were exposed to erlotinib and C . acnes for 6 hours and IL36G levels were analyzed by qPCR. Data represent mean ± SEM ( n = 3). ( D ) Keratinocyte cell lines in which KLF4 was knocked out by CRISPR/Cas9 were exposed to trametinib (2 μg/mL) for 24 hours and total cell lysates were collected for Western blotting with antibodies against KLF4 and β-actin. The cells were exposed to trametinib for 24 hours and isolated RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). All blots were run contemporaneously with the same protein samples. Data are representative of 3 independent experiments. ( E ) Mutations generated by CRISPR/Cas9 in the KLF4 binding site. Red nucleotides are the PAM sequence and blue nucleotides hybridize to the sgRNA. KLF4 binding site–mutant cells were exposed to trametinib and Pam3CSK4 for 24 hours. Total RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). Data were analyzed with 1-way ANOVA followed by Dunnett’s ( B and E ) or Tukey’s multiple-comparisons test ( C ) or with 2-tailed unpaired t test ( D ). ** P

    Article Snippet: Keratinocytes were grown in keratinocyte serum-free medium (17005-042, Thermo Fisher Scientific), supplemented with EGF and bovine pituitary extract (BPE) (Thermo Fisher Scientific), and seeded for experiments after 3 passages.

    Techniques: Activity Assay, Inhibition, Dominant Negative Mutation, Western Blot, Real-time Polymerase Chain Reaction, CRISPR, Isolation, Generated, Binding Assay, Sequencing, Mutagenesis

    Blockade of the EGFR/MEK/ERK pathway increases keratinocyte expression of KLF4. ( A ) Quantitative PCR was performed to evaluate gene expression in RNA isolated from biopsies of 4 patients with acneiform eruption and 10 healthy control (HC) skin biopsies. Data represent mean ± SD. ( B ) PHKs were pre-exposed to the BRAF inhibitor vemurafenib (1 μg/mL) for 30 minutes and exposed to the MEK inhibitor trametinib (2 μg/mL) and C . acnes (MOI of 10) for 6 hours. Data represent mean ± SEM ( n = 3). Data were analyzed with 2-tailed unpaired t test ( A ) or 1-way ANOVA followed by Tukey’s multiple-comparisons test ( B ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

    doi: 10.1172/JCI128678

    Figure Lengend Snippet: Blockade of the EGFR/MEK/ERK pathway increases keratinocyte expression of KLF4. ( A ) Quantitative PCR was performed to evaluate gene expression in RNA isolated from biopsies of 4 patients with acneiform eruption and 10 healthy control (HC) skin biopsies. Data represent mean ± SD. ( B ) PHKs were pre-exposed to the BRAF inhibitor vemurafenib (1 μg/mL) for 30 minutes and exposed to the MEK inhibitor trametinib (2 μg/mL) and C . acnes (MOI of 10) for 6 hours. Data represent mean ± SEM ( n = 3). Data were analyzed with 2-tailed unpaired t test ( A ) or 1-way ANOVA followed by Tukey’s multiple-comparisons test ( B ). * P

    Article Snippet: Keratinocytes were grown in keratinocyte serum-free medium (17005-042, Thermo Fisher Scientific), supplemented with EGF and bovine pituitary extract (BPE) (Thermo Fisher Scientific), and seeded for experiments after 3 passages.

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

    Increased production of IL-36γ in primary keratinocytes and lesional skin of patients suffering from acneiform eruptions in response to EGFR inhibition and C. acnes . ( A ) Gene expression profiling from lesional skin of 5 patients and 5 healthy controls (HC). Heatmap of the top 12 most differentially expressed genes ranked from lowest false discovery rate (FDR) and 12 selected genes are shown. ( B ) Quantitative PCR (qPCR) of mRNA from lesional skin samples of 10 EGFR inhibitor–treated patients with acneiform eruption and 10 healthy control skin biopsies. Data represent mean ± SD. ( C ) Immunohistochemical staining with goat anti–IL-36γ antibody of formalin-fixed, paraffin-embedded skin sections of acneiform eruption patient and normal donors. Scale bars: 100 μm. Pictures are representative of 5 patients and 5 healthy individuals. ( D ) PHKs were exposed to erlotinib (EGFR inhibitor, 1 μM) and C . acnes (MOI of 10) for 6 hours. Total RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). ( E ) PHKs were exposed to erlotinib (1 μM) or C . acnes (MOI of 10) or both for 24 hours. Cell lysates were analyzed by Western blotting using specific antibodies against IL-36γ and β-actin. Blots were run contemporaneously with the same protein samples. ( F ) PHKs were exposed to erlotinib (1 μM) and Pam3CSK4 (5 μg/mL). IL-36γ secretion was measured by ELISA in culture supernatants. Data represent mean ± SEM ( n = 3). ( G ) Ex vivo skin explants were exposed to erlotinib (1 μM), Pam3CSK4 (5 μg/mL), and/or human IL-36Ra (1 μg/mL). The skin samples were then analyzed by qPCR. Data represent mean ± SEM ( n = 4). Data were analyzed with 2-tailed unpaired t test ( B ), and 1-way ANOVA followed by Dunnett’s ( D and F ) or Tukey’s multiple-comparisons test ( G ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

    doi: 10.1172/JCI128678

    Figure Lengend Snippet: Increased production of IL-36γ in primary keratinocytes and lesional skin of patients suffering from acneiform eruptions in response to EGFR inhibition and C. acnes . ( A ) Gene expression profiling from lesional skin of 5 patients and 5 healthy controls (HC). Heatmap of the top 12 most differentially expressed genes ranked from lowest false discovery rate (FDR) and 12 selected genes are shown. ( B ) Quantitative PCR (qPCR) of mRNA from lesional skin samples of 10 EGFR inhibitor–treated patients with acneiform eruption and 10 healthy control skin biopsies. Data represent mean ± SD. ( C ) Immunohistochemical staining with goat anti–IL-36γ antibody of formalin-fixed, paraffin-embedded skin sections of acneiform eruption patient and normal donors. Scale bars: 100 μm. Pictures are representative of 5 patients and 5 healthy individuals. ( D ) PHKs were exposed to erlotinib (EGFR inhibitor, 1 μM) and C . acnes (MOI of 10) for 6 hours. Total RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). ( E ) PHKs were exposed to erlotinib (1 μM) or C . acnes (MOI of 10) or both for 24 hours. Cell lysates were analyzed by Western blotting using specific antibodies against IL-36γ and β-actin. Blots were run contemporaneously with the same protein samples. ( F ) PHKs were exposed to erlotinib (1 μM) and Pam3CSK4 (5 μg/mL). IL-36γ secretion was measured by ELISA in culture supernatants. Data represent mean ± SEM ( n = 3). ( G ) Ex vivo skin explants were exposed to erlotinib (1 μM), Pam3CSK4 (5 μg/mL), and/or human IL-36Ra (1 μg/mL). The skin samples were then analyzed by qPCR. Data represent mean ± SEM ( n = 4). Data were analyzed with 2-tailed unpaired t test ( B ), and 1-way ANOVA followed by Dunnett’s ( D and F ) or Tukey’s multiple-comparisons test ( G ). * P

    Article Snippet: Keratinocytes were grown in keratinocyte serum-free medium (17005-042, Thermo Fisher Scientific), supplemented with EGF and bovine pituitary extract (BPE) (Thermo Fisher Scientific), and seeded for experiments after 3 passages.

    Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded, Western Blot, Enzyme-linked Immunosorbent Assay, Ex Vivo

    CD147 inhibition impairs galectin-3-mediated secretion of MMP9. (A) Human corneal keratinocytes transfected with either non-targeting siRNA (siScramble) or CD147 siRNA (siCD147) were exposed to serum-free medium alone (NT), rGal-3 or rGal-3C for 4 h.

    Journal: Journal of Cell Science

    Article Title: Molecular basis for MMP9 induction and disruption of epithelial cell–cell contacts by galectin-3

    doi: 10.1242/jcs.148510

    Figure Lengend Snippet: CD147 inhibition impairs galectin-3-mediated secretion of MMP9. (A) Human corneal keratinocytes transfected with either non-targeting siRNA (siScramble) or CD147 siRNA (siCD147) were exposed to serum-free medium alone (NT), rGal-3 or rGal-3C for 4 h.

    Article Snippet: Briefly, cells were grown in keratinocyte serum-free medium (K-SFM) (Life Technologies, Carlsbad, CA, USA) to confluence (monolayer).

    Techniques: Inhibition, Transfection

    Effects of a consortium of oral streptococci on keratinocytes. Human oral keratinocytes were grown on control surfaces (C) in the absence (left panel) or presence (right panel) of a consortium of oral streptococci. The scale bar represents 50 μm.

    Journal: BMC Oral Health

    Article Title: Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces

    doi: 10.1186/1472-6831-14-75

    Figure Lengend Snippet: Effects of a consortium of oral streptococci on keratinocytes. Human oral keratinocytes were grown on control surfaces (C) in the absence (left panel) or presence (right panel) of a consortium of oral streptococci. The scale bar represents 50 μm.

    Article Snippet: Human oral keratinocytes Immortalized normal human oral keratinocytes (OKF6/TERT-2) [ ] obtained from Dr James Rheinwald (Brigham and Women’s Hospital, Boston, USA), were seeded into culture dishes in serum-free keratinocyte medium (Gibco) supplemented with 0.2 ng ml-1 human recombinant epidermal growth factor, 25 μg bovine pituitary extract ml-1 and 0.3 mM CaCl2 containing 1 IU penicillin ml-1 and 1 μg streptomycin ml-1 (DF-K medium) and cultured in 5% CO2 in air at 37°C.

    Techniques:

    The blockage of mediator subunits resulted in hyperproliferation of keratinocytes Keratinocytes were transfected with small interfering RNA (siRNA) to block expression of vitamin D receptor (VDR) or mediator subunits. Silencing of VDR, MED1, MED21 resulted in morphological changes ( a , phase contrast). Bar = 20 μm. Proliferation was accessed by BrdU incorporation. BrdU was immunohistochemically visualized ( a , BrdU staining; bars = 20μm), and the ratio of BrdU-positive cells (brown) per total cells (blue nuclear counter staining) was calculated using Bioquant (Materials and Methods) ( b ). The data are shown as mean ± SD of three different preparations of keratinocytes. Statistical significance evaluated by t -test (** P

    Journal: The Journal of investigative dermatology

    Article Title: The Transcriptional Coactivator DRIP/Mediator Complex Is Involved in Vitamin D Receptor Function and Regulates Keratinocyte Proliferation and Differentiation

    doi: 10.1038/jid.2010.148

    Figure Lengend Snippet: The blockage of mediator subunits resulted in hyperproliferation of keratinocytes Keratinocytes were transfected with small interfering RNA (siRNA) to block expression of vitamin D receptor (VDR) or mediator subunits. Silencing of VDR, MED1, MED21 resulted in morphological changes ( a , phase contrast). Bar = 20 μm. Proliferation was accessed by BrdU incorporation. BrdU was immunohistochemically visualized ( a , BrdU staining; bars = 20μm), and the ratio of BrdU-positive cells (brown) per total cells (blue nuclear counter staining) was calculated using Bioquant (Materials and Methods) ( b ). The data are shown as mean ± SD of three different preparations of keratinocytes. Statistical significance evaluated by t -test (** P

    Article Snippet: Normal epidermal keratinocytes were isolated from neonatal-human foreskin and grown in serum-free keratinocyte growth medium (Cascade Biologics, Portland OR) as previously described ( ).

    Techniques: Transfection, Small Interfering RNA, Blocking Assay, Expressing, BrdU Incorporation Assay, BrdU Staining, Staining

    The mediator complex is involved in regulation of Wnt target genes Keratinocytes were transfected with small interfering RNA (siRNA) for vitamin D receptor (VDR) or mediators and treated with either vehicle (EtOH; open bars) or 1,25(OH) 2 D 3 (closed bars). The mRNA levels of cyclin D1, glioma-associated oncogene homolog (Gli 1), peptidyl arginine deiminase 1 (PADI1), and the control gene L19 were measured. To normalize the data in each experiment, the percentage of induction compared with sicontrol was calculated. The data are shown as mean±SD of three different preparations of keratinocytes. Statistical significance (* P

    Journal: The Journal of investigative dermatology

    Article Title: The Transcriptional Coactivator DRIP/Mediator Complex Is Involved in Vitamin D Receptor Function and Regulates Keratinocyte Proliferation and Differentiation

    doi: 10.1038/jid.2010.148

    Figure Lengend Snippet: The mediator complex is involved in regulation of Wnt target genes Keratinocytes were transfected with small interfering RNA (siRNA) for vitamin D receptor (VDR) or mediators and treated with either vehicle (EtOH; open bars) or 1,25(OH) 2 D 3 (closed bars). The mRNA levels of cyclin D1, glioma-associated oncogene homolog (Gli 1), peptidyl arginine deiminase 1 (PADI1), and the control gene L19 were measured. To normalize the data in each experiment, the percentage of induction compared with sicontrol was calculated. The data are shown as mean±SD of three different preparations of keratinocytes. Statistical significance (* P

    Article Snippet: Normal epidermal keratinocytes were isolated from neonatal-human foreskin and grown in serum-free keratinocyte growth medium (Cascade Biologics, Portland OR) as previously described ( ).

    Techniques: Transfection, Small Interfering RNA

    The function of MED1 and MED21 in calcium-induced E-cadherin translocation Keratinocytes were transfected with small interfering RNA (siRNA) to block expression of vitamin D receptor (VDR) or mediator subunits, and treated with calcium for 5 minutes to initiate keratinocyte adhesion. Translocation of E-cadherin (green) and β-catenin (red) to the membrane was visualized by immunofluorescence (green and red images were superimposed, so that sites of overlap are visualized as yellow) ( a ). Bars =20 μm. Calcium-induced E-cadherin translocation to the plasma membrane ( b , western analysis, upper lanes) compared with that in total lysates ( b , lower lanes). The mRNA expression of calcium-sensing receptor (CaR; c , left panel) and the calcium-induced translocation of CaR to the membrane ( c , right panel) are also shown. CaR protein bands in sicontrol (lane 5, 6) were not run consecutively with one in siVDR and si vitamin D receptor interacting proteins (DRIP) (lane 1–4), but consistently appeared in this pattern.

    Journal: The Journal of investigative dermatology

    Article Title: The Transcriptional Coactivator DRIP/Mediator Complex Is Involved in Vitamin D Receptor Function and Regulates Keratinocyte Proliferation and Differentiation

    doi: 10.1038/jid.2010.148

    Figure Lengend Snippet: The function of MED1 and MED21 in calcium-induced E-cadherin translocation Keratinocytes were transfected with small interfering RNA (siRNA) to block expression of vitamin D receptor (VDR) or mediator subunits, and treated with calcium for 5 minutes to initiate keratinocyte adhesion. Translocation of E-cadherin (green) and β-catenin (red) to the membrane was visualized by immunofluorescence (green and red images were superimposed, so that sites of overlap are visualized as yellow) ( a ). Bars =20 μm. Calcium-induced E-cadherin translocation to the plasma membrane ( b , western analysis, upper lanes) compared with that in total lysates ( b , lower lanes). The mRNA expression of calcium-sensing receptor (CaR; c , left panel) and the calcium-induced translocation of CaR to the membrane ( c , right panel) are also shown. CaR protein bands in sicontrol (lane 5, 6) were not run consecutively with one in siVDR and si vitamin D receptor interacting proteins (DRIP) (lane 1–4), but consistently appeared in this pattern.

    Article Snippet: Normal epidermal keratinocytes were isolated from neonatal-human foreskin and grown in serum-free keratinocyte growth medium (Cascade Biologics, Portland OR) as previously described ( ).

    Techniques: Translocation Assay, Transfection, Small Interfering RNA, Blocking Assay, Expressing, Immunofluorescence, Western Blot

    Tonsillar explant culture scheme. Cultures were grown as described in Materials and Methods. The procedure involves three separate steps. In step 1, tonsillar explants are placed beneath tissue culture transwell inserts. The membrane is porous, and a weight is placed on top of the insert membrane. DMEM-F12 containing 10% FBS and 1 mM Ca 2+ was used during the first 4 days of culture. In step 2, the inserts are removed and the culture medium is changed to a low-calcium defined serum-free medium with keratinocyte growth factor (Gibco). This ensures the outgrowth of epithelial cells, abolishes fibroblast growth, and kills lymphocytes. In step 3, the explants are removed and epithelial cells are transferred to new dishes and culture slides. Photographs of the cultures at different stages are shown. Epithelial cells begin to migrate out of the tissue explants (T) on day 4 (d4) (black arrow). On day 7, large colonies surround the tissue explants with no visible indication of surviving lymphocytes. On day 7, large colonies ( > 1 cm in diameter) of cells with typical epithelial morphology surround the explants. By day 10, adherent cells have different morphologies, some cells having acquired a very large cytoplasm-to-nucleus ratio (white arrow), often seen in differentiated squamous epithelial cells. Thus, it appears that an active epithelial differentiation process is ongoing in the cultures.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Infection in Ex Vivo Tonsil Epithelial Cell Cultures of Asymptomatic Carriers

    doi: 10.1128/JVI.78.22.12613-12624.2004

    Figure Lengend Snippet: Tonsillar explant culture scheme. Cultures were grown as described in Materials and Methods. The procedure involves three separate steps. In step 1, tonsillar explants are placed beneath tissue culture transwell inserts. The membrane is porous, and a weight is placed on top of the insert membrane. DMEM-F12 containing 10% FBS and 1 mM Ca 2+ was used during the first 4 days of culture. In step 2, the inserts are removed and the culture medium is changed to a low-calcium defined serum-free medium with keratinocyte growth factor (Gibco). This ensures the outgrowth of epithelial cells, abolishes fibroblast growth, and kills lymphocytes. In step 3, the explants are removed and epithelial cells are transferred to new dishes and culture slides. Photographs of the cultures at different stages are shown. Epithelial cells begin to migrate out of the tissue explants (T) on day 4 (d4) (black arrow). On day 7, large colonies surround the tissue explants with no visible indication of surviving lymphocytes. On day 7, large colonies ( > 1 cm in diameter) of cells with typical epithelial morphology surround the explants. By day 10, adherent cells have different morphologies, some cells having acquired a very large cytoplasm-to-nucleus ratio (white arrow), often seen in differentiated squamous epithelial cells. Thus, it appears that an active epithelial differentiation process is ongoing in the cultures.

    Article Snippet: The medium was replaced by defined keratinocyte serum-free medium (SFM) plus keratinoctyte growth factor (KGF) (Invitrogen) containing 0.1 mM Ca.

    Techniques:

    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

    Keratinocytes and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for

    Journal:

    Article Title: Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion

    doi: 10.1128/JVI.79.12.7363-7370.2005

    Figure Lengend Snippet: Keratinocytes and HMVEC-d have distinct patterns of cytokine production following vaccinia virus infection. Keratinocytes and HMVEC-d were infected with WR-VV at an MOI of 10 and cultured for 20 h. The supernatant samples were collected and analyzed for

    Article Snippet: Gibco keratinocyte-sfm culture medium was supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml epidermal growth factor (EGF; Invitrogen, Grand Island, NY) plus 0.3 mM CaCl2 and antibiotics ( ).

    Techniques: Infection, Cell Culture

    VV undergoes only limited replication in keratinocytes, while replicating robustly in dermal fibroblasts and endothelial cells. Primary cultures of the indicated skin cells were infected with WR-VV at an MOI of 0.1 (0.1 PFU per cell). Sixty hours later,

    Journal:

    Article Title: Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion

    doi: 10.1128/JVI.79.12.7363-7370.2005

    Figure Lengend Snippet: VV undergoes only limited replication in keratinocytes, while replicating robustly in dermal fibroblasts and endothelial cells. Primary cultures of the indicated skin cells were infected with WR-VV at an MOI of 0.1 (0.1 PFU per cell). Sixty hours later,

    Article Snippet: Gibco keratinocyte-sfm culture medium was supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml epidermal growth factor (EGF; Invitrogen, Grand Island, NY) plus 0.3 mM CaCl2 and antibiotics ( ).

    Techniques: Infection

    Keratinocytes and HMVEC-d show distinct patterns of cytokine production following vaccinia virus infection.

    Journal:

    Article Title: Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion

    doi: 10.1128/JVI.79.12.7363-7370.2005

    Figure Lengend Snippet: Keratinocytes and HMVEC-d show distinct patterns of cytokine production following vaccinia virus infection.

    Article Snippet: Gibco keratinocyte-sfm culture medium was supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml epidermal growth factor (EGF; Invitrogen, Grand Island, NY) plus 0.3 mM CaCl2 and antibiotics ( ).

    Techniques: Infection

    VV-infected keratinocytes do not secrete soluble factors that can inhibit secondary viral infection. Keratinocytes were infected with WR-VV at an MOI of 0.1 (low dose) or 10 (high dose) for 24 h. Supernatant samples were harvested and stored at −80°C.

    Journal:

    Article Title: Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion

    doi: 10.1128/JVI.79.12.7363-7370.2005

    Figure Lengend Snippet: VV-infected keratinocytes do not secrete soluble factors that can inhibit secondary viral infection. Keratinocytes were infected with WR-VV at an MOI of 0.1 (low dose) or 10 (high dose) for 24 h. Supernatant samples were harvested and stored at −80°C.

    Article Snippet: Gibco keratinocyte-sfm culture medium was supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml epidermal growth factor (EGF; Invitrogen, Grand Island, NY) plus 0.3 mM CaCl2 and antibiotics ( ).

    Techniques: Infection

    The limited VV replication in keratinocytes is not due to poor cell-to-cell transmission. Known numbers of the indicated skin cells were infected with WR-VV at an MOI of 10 (10 PFU/cell). Sixty hours later, the numbers of virus particles in the cultures

    Journal:

    Article Title: Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion

    doi: 10.1128/JVI.79.12.7363-7370.2005

    Figure Lengend Snippet: The limited VV replication in keratinocytes is not due to poor cell-to-cell transmission. Known numbers of the indicated skin cells were infected with WR-VV at an MOI of 10 (10 PFU/cell). Sixty hours later, the numbers of virus particles in the cultures

    Article Snippet: Gibco keratinocyte-sfm culture medium was supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml epidermal growth factor (EGF; Invitrogen, Grand Island, NY) plus 0.3 mM CaCl2 and antibiotics ( ).

    Techniques: Transmission Assay, Infection

    Keratinocytes and HMVEC-d show distinct patterns of cytokine production following vaccinia virus infection.

    Journal:

    Article Title: Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion

    doi: 10.1128/JVI.79.12.7363-7370.2005

    Figure Lengend Snippet: Keratinocytes and HMVEC-d show distinct patterns of cytokine production following vaccinia virus infection.

    Article Snippet: Gibco keratinocyte-sfm culture medium was supplemented with 25 μg/ml bovine pituitary extract and 0.2 ng/ml epidermal growth factor (EGF; Invitrogen, Grand Island, NY) plus 0.3 mM CaCl2 and antibiotics ( ).

    Techniques: Infection

    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