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: International Journal of Molecular Sciences

Article Title: Cell-Penetrating Delivery of Nitric Oxide by Biocompatible Dinitrosyl Iron Complex and Its Dermato-Physiological Implications

doi: 10.3390/ijms221810101

Figure Lengend Snippet: Cell viability assay of the ( a ) mouse skin melanoma (B16-F10) and ( b ) human skin keratinocyte cells (HaCaT) cells, respectively, treated with different concentrations of DNIC-2 for 24 h **** p < 0.001 compared to the group without treatment of DNIC-2 . ( c ) Cell viability assay of the reconstructed human epidermis (RhE) model treated with PBS, 5% SDS, and 50 μM of DNIC-2 , respectively. ( d ) Cell viability assay of the reconstructed human cornea-like epithelium model treated with DPBS, methyl acetate, and 50 μM of DNIC-2 , respectively.

Article Snippet: CCD-966Sk human skin fibroblasts and B16-F10 mouse skin melanoma cells were purchased from Bioresource Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan), whereas human skin keratinocyte cell line, HaCaT, was purchased from Elabscience Biotechnology Inc. (Elabscience ® EP-CL-0090, Houston, TX, USA).

Techniques: Viability Assay

Journal: Frontiers in Pharmacology

Article Title: Syzygium aqueum : A Polyphenol- Rich Leaf Extract Exhibits Antioxidant, Hepatoprotective, Pain-Killing and Anti-inflammatory Activities in Animal Models

doi: 10.3389/fphar.2018.00566

Figure Lengend Snippet: Effect of Syzygium aqueum extracts on human keratinocytes. (A) Biocompatibility of S. aqueum extract in dose (25–200 μg/mL) and time (24–48 h) manner. Survival rate of the cells was assessed by the MTT assay. (B) ROS production in cells treated or untreated with theextract (100 μg/mL) and then irradiated by 100 J/cm 2 UVA (+) or not (–). (C) Total GSH levels determined by DTNB assay. Results expressed as mean ± SD of three assays. Significant ∗ p < 0.01; ∗∗ p < 0.001 compared to untreated control. Analysis performed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. (D) Immunoblotting bands of the oxidative stress activated protein, p38. An equal amount (100 μg) of total lysate from each sample was resolved by SDS-PAGE with GAPDH as a control.

Article Snippet: Human epidermal keratinocytes (HaCaT), provided by Innoprot (Biscay, Spain), were cultured as described in .

Techniques: MTT Assay, Irradiation, DTNB Assay, Western Blot, SDS Page

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