Journal: Toxicology and applied pharmacology

Article Title: Monoubiquitinated γ-H2AX: abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

doi: 10.1016/j.taap.2018.07.007

Figure Lengend Snippet: Panels A-C used antibodies #1. (A) Impact of gel polyacrylamide percentage and transfer conditions to PVDF membranes. Treatments of H460 cells: 6 h with 3 mM hydroxyurea (HU), 6 or 16 h with 5 μM aphidicolin (Aphi) or 1 h with 5 μM bleomycin (Bleo). All γ-H2AX images had 5 s film exposures. (B) Ethanol effect in semi-dry transfer. IMR90 cells were treated with 40 μM etoposide (Eto) for 1 h. (C) Comparison of wet (pH 8.2 and pH 8.8) and semi-dry transfers using 12% ethanol-containing buffers (10 s film exposures for all γ-H2AX images). IMR90 cells were treated with 40 μM etoposide for 1 h. (D) Effectiveness of different antibodies using semi-dry transfer with 12% ethanol. Normal human keratinocytes were treated for 1 h with 30 μM etoposide or 0.5 μM bleomycin. Antibodies #1–3: 1:1000 dilutions and 5 s exposures, antibody #4: 1:1000 dilution and 60 s exposure. (E) Comparison of antibodies #1 and #5 (both at 1:1000 dilutions) in H460 cells treated with bleomycin for 1 h. (F) γ-H2AX blot (Ab#1) in control and H2AX-depleted IMR90 cells treated with 100 μM etoposide for 1h.

Article Snippet: IMR90, WI38, normal human neonatal keratinocytes (PCS-200–010), H460, A549, U2OS, mouse embryonic fibroblasts (MEFs, SCRC-1040), telomerase-immortalized human bronchial epithelial HBEC3 (CRL-4051), Daudi, HCT116, Ramos, Raji and CA46 were obtained from ATCC.

Techniques: Comparison, Control

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Changes in mRNA expression for P2Y receptors induced by ATRA in NHEKs. Diagram shows the percentage of the quantity after amplification by real-time RT-PCR for P2Y1, P2Y2 and P2Y11 receptor mRNAs extracted from NHEKs treated with 1 µM ATRA for 2 h. Asterisks show significant difference from control groups ( white columns , ** P < 0.01). mRNAs of P2Y2 receptors were increased by more than twofold vs. control. Data were obtained from at least three independent experiments.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Expressing, Amplification, Quantitative RT-PCR, Control

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Time- and concentration-dependency of three different retinoids-induced changes in mRNAs in NHEKs. Diagram shows the quantity of P2Y2 mRNAs detected by real-time RT-PCR after treatment with 0.001–1 µM ATRA (A), Am80 (B) and 9- cis RA (C) for 2–24 h. The P2Y2 mRNA levels in cells treated with various concentrations of retinoids were normalized by those in retinoids-untreated control cells at each incubation period (2, 6, 12 and 24 h), and expressed as “percentage (%) of control.” All these retinoids, and especially Am80, caused a linear increase in P2Y2 mRNAs in a concentration- and time-dependent fashion. Asterisks show significant difference in the P2Y2 mRNA levels from control groups ( * P < 0.05; ** P < 0.01). Data were obtained from at least three independent experiments.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Concentration Assay, Quantitative RT-PCR, Control, Incubation

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Enhancement by ATRA and Am80 of P2Y2 receptor-mediated increase in [Ca 2+ ]i in NHEKs. A. Typical traces of the UTP-evoked changes in [Ca 2+ ]i in NHEKs. NHEKs were incubated with 0.1 µM ATRA ( middle ) or Am80 ( bottom ) for 6 h, incubated with normal culture medium for another 18 h, and then the fura-2 based [Ca 2+ ]i measurement was performed. UTP (100 µM) was applied to cells for 10 s and the increase in the ΔF340/F380 ratio was calculated ( n = 110–125). After the initial UTP-application, the extracellular Ca 2+ was removed (0 Ca 2+ ), and the second UTP was applied to the cells in the absence of extracellular Ca 2+ . Effect of ATRA and Am80 on the UTP-evoked elevation in [Ca 2+ ]i in NHEKs in the presence and absence of extracellular Ca 2+ was summarized in B. Asterisks show significant difference from control (without retinoids) ( * P < 0.05; ** P < 0.01).

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Incubation, Control

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Visualization of release of ATP from NHEKs. The image panels in A show ATP-derived photons ( white dots ) in a field of ATRA-treated ( right ) and -untreated control NHEKs ( left ). NHEKs were incubated with 0.1 µM ATRA for 6 h. Cells were bathed in luciferin-luciferase reagent and the bioluminescence signals were obtained with a VIM camera (see Materials and methods Section) with an exposure time of 10 s. Sequential images show the ATP-derived photon-signals before (−10 s; b & b′) and 10 (c & c′), 20 (d & d′) and 30 s (e & e′) after mechanical stimulation. The positions of the pipettes are shown in phasecontrast images of NHEKs (a & a′). In B, the accumulative photon intensity in 60 s was converted to the absolute extracellular ATP concentration using a standard ATP-photon intensity relationship curve determined with an ATP standard solution (control, n = 17; ATRA-treated, n = 14). Photons within 50 mms squares around the stimulated site (shown as white squares in a & a′ panels in A) were calculated. Asterisks show significant difference from basal groups ( ** P < 0.01) and dagger shows significant difference from control basal groups († P < 0.05).

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Derivative Assay, Control, Incubation, Luciferase, Concentration Assay

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: ATRA-induced changes in expression pattern of P2 receptors in NHEKs.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Expressing, Control

Journal: Pharmaceutics

Article Title: Tissue Engineered Human Skin Equivalents

doi: 10.3390/pharmaceutics4010026

Figure Lengend Snippet: Summary of commercially available human skin equivalents.

Article Snippet: Epiderm , MatTek , , Neonatal human-derived epidermal keratinocytes (NHEK) cultured to form a multi-layered, highly differentiated model of the human epidermis.

Techniques: Ex Vivo, Cell Culture, In Vitro, Functional Assay

Journal: Cytotechnology

Article Title: Restoration of calcium-induced differentiation potential and tight junction formation in HaCaT keratinocytes by functional attenuation of overexpressed high mobility group box-1 protein

doi: 10.1007/s10616-019-00367-6

Figure Lengend Snippet: HaCaT cells express considerable amount of a proinflammatory mediator HMGB1. a Expression of HMGB1 in NHEK and HaCaT cells. Compare to primary cultured keratinocyte, HaCaT cells actively produce and secrete HMGB1 in Supernatant, concentrated conditioned medium. Experiments were repeated three times and the representative blot images were shown. b Immunodetection of expression and distribution of HMGB1. HGM1 was detectable in both the nuclei and cytosol (arrows) of HaCaT cells. Experiments were repeated three times and the representative images were shown. Bars, 25 μm. c Effect of GK2 on the mRNA expression of interferon-β (IFNβ), one of the most sensitive HMGB1 downstream elements in keratinocytes. All the independent experiments (exp1, 2 and 3) demonstrated clear down-regulation of IFNβ by the treatment with GΚ2, albeit its expression was varied depending on the culture

Article Snippet: The normal human epidermal keratinocytes (NHEK) obtained from Kurabo LTD were maintained in HuMedia-KG2 medium with growth supplement as instructed in the protocol (Kurabo), and those in low passage number (less than four) were used in this study.

Techniques: Expressing, Cell Culture, Immunodetection

Journal: Cytotechnology

Article Title: Restoration of calcium-induced differentiation potential and tight junction formation in HaCaT keratinocytes by functional attenuation of overexpressed high mobility group box-1 protein

doi: 10.1007/s10616-019-00367-6

Figure Lengend Snippet: Distinct effects of SP600125 and GK2 on TJ formation in HaCaT cells. a upper, Accumulation of a TJ component ZO-1 (red) in cells with SP600125 and GK2 on day 1 (left) and day3 (right). The nuclei were counterstained with DAPI. Bars, 25 μm. Lower, quantification of relative number of TJs on day 1 and 3. Average numbers of TJs from three line scan assays (n = 3 for each category, as shown in Figs. 1 and ​and3)3) were calculated and relative numbers to those of day1 are shown. TJs induced by SP600125 appeared to be stable, whereas those by GK2 were almost disrupted on day 3. Bars, 50 μm. b Comparative diagram of TJ-formed cell populations in HaCaT keratinocytes treated with SP600125 and GK2. Cells treated with GK2 construct double cell layers and only upper cells formed TJs (see Figs. 1 and ​and3).3). c Schema of the effect of GK2 on HaCaT cells that produce an inflammatory mediator HMGB1. GK2 abrogates the inflammatory action of HMGB1, leading to restoration of the differentiation potential and of the TJ formation program

Article Snippet: The normal human epidermal keratinocytes (NHEK) obtained from Kurabo LTD were maintained in HuMedia-KG2 medium with growth supplement as instructed in the protocol (Kurabo), and those in low passage number (less than four) were used in this study.

Techniques: Construct

Journal: PLoS ONE

Article Title: Impact of the Genome on the Epigenome Is Manifested in DNA Methylation Patterns of Imprinted Regions in Monozygotic and Dizygotic Twins

doi: 10.1371/journal.pone.0025590

Figure Lengend Snippet: (A) The Position Weight Matrix (PWM) of the CTCF consensus sequence as determined by Cuddapah et al. (2009) harbours a strong preference for a cytosine residue at position 6 of the PWM. Within the region upstream the H19 gene, this cytosine is present in CTCF binding site numbers 4 and 6 (for the rs10732516 [G] allele) . The CTCF binding site number 5 within the H19 region harbours a thymidine at position 6, and is identical in sequence to CTCF binding site number 6 for the rs10732516 [A] allele (CTCF sequences are on the minus strand). (B) Analysis of CTCF binding at the H19 promoter region covering CTCF binding sites 4, 5 and 6 in six normal cell lines identified site number 5 to have minimal binding affinity within the region (ChIP-seq data from the ENCODE Chromatin Group at the Broad Institute ; UCSC Genome Browser; HMEC: human mammary epithelial cells; HSMM: normal human skeletal muscle myoblasts; HUVEC: human umbilical vein endothelial cells; NHEK: normal human epidermal keratinocytes; NHLF: normal human lung fibroblasts; H1ES: human embryonic stem cell line H1). (C) Effect of cytosine versus thymidine at position 6 of the PWM on the average enrichment score near CTCF sites within the genome of HMEC cells. Positions matching the PWM with 80% or greater sequence identity were identified and the average enrichment scores near these sites were determined −500 to +500 base pairs relative to the CTCF motif. Results for the other cell lines can be found as Figure S8. These combined results suggest a strong influence of the rs10732516 genotype on the binding affinity of CTCF at this position in the H19 -ICR.

Article Snippet: Publically available ChIP-seq data from the ENCODE Chromatin Group at the Broad Institute was downloaded for all available normal cell lines (HMEC [human mammary epithelial cells], HSMM [normal human skeletal muscle myoblasts], HUVEC [human umbilical vein endothelial cells], NHEK [normal human epidermal keratinocytes], NHLF [normal human lung fibroblasts] and the H1 human embryonic stem cell line).

Techniques: Sequencing, Residue, Binding Assay, ChIP-sequencing