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