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: PLoS ONE

Article Title: ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

doi: 10.1371/journal.pone.0105498

Figure Lengend Snippet: ( a ) Immunofluorescence labeling confocal microscopy to localize TSLPR and IL-7Rα in the area of epidermis where ΔNp63 was not detected. Bar = 20 µm. ( b , c ) Quantitative RT-PCR ( b ) and immunoblot analysis ( c ) against ΔNp63, TSLPR, IL-7Rα and TSLP in control siRNA (siControl) and p63-specific siRNA (sip63)-transfected HaCaT keratinocytes. β-actin was used as a loading control. The cells were harvested for assays at 72 hr after siRNA transfection. Student's t test. ** P <0.01 and N.S., not significant. ( d ) Immunofluorescence labeling fluorescence microscopy to localize TSLPR in siControl and sip63 HaCaT keratinocytes. The cells were fixed at 72 hr after siRNA introduction. DAPI, 4′,6-diamidino-2-phenylindole. Bar = 50 µm. Data are representative of at least three independent experiments.

Article Snippet: Human primary keratinocytes were purchased from DS Pharma Biomedical (Tokyo, Japan) and cultured in a serum-free medium for human keratinocytes (DS Pharma Biomedical).

Techniques: Immunofluorescence, Labeling, Confocal Microscopy, Quantitative RT-PCR, Western Blot, Control, Transfection, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

doi: 10.1371/journal.pone.0105498

Figure Lengend Snippet: ( a ) Quantitative RT-PCR demonstrating the levels of ΔNp63, p73, TSLP, TSLPR and IL-7Rα mRNAs at 24 hr after treatment with 25 µg/ml poly I:C. One-way ANOVA followed by Tukey's multiple-comparison test. *** P <0.005 and **** P <0.001 versus control. ( b ) Immunoblot analysis showing the level of ΔNp63 at different time course after treatment with 25 µg/ml poly I:C. ( c ) Phase-contrast microscopy demonstrating the morphological changes of primary keratinocytes at 24 hr after 25 µg/ml poly I:C treatment. Bar = 50 µm. Data are representative of three independent experiments.

Article Snippet: Human primary keratinocytes were purchased from DS Pharma Biomedical (Tokyo, Japan) and cultured in a serum-free medium for human keratinocytes (DS Pharma Biomedical).

Techniques: Quantitative RT-PCR, Comparison, Control, Western Blot, Microscopy

Journal: PLoS ONE

Article Title: ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

doi: 10.1371/journal.pone.0105498

Figure Lengend Snippet: ( a, b ) Quantitative RT-PCR ( a ) and ELISA ( b ) demonstrate the level of endogenous TSLP at 24 hr ( a ) or 72 hr ( b ) after treatment with exogenous TSLP (1 or 10 ng/ml) under the condition of TLR3 stimulation (5 µg/ml poly I:C) in primary keratinocytes. ( c, d ) Quantitative RT-PCR ( c ) and ELISA ( d ) show the level of endogenous TSLP at 24 hr ( c ) or 72 hr ( d ) after stimulation with 10 µg/ml poly I:C and 10 ng/ml TSLP in siControl and sip63 HaCaT keratinocytes. ( e ) Quantitative RT-PCR confirming whether neutralization of TSLP inhibits the generation of TSLP under the condition of TLR3 stimulation (5 µg/ml poly I:C) in primary keratinocytes. One-way ANOVA followed by Tukey's multiple-comparison test. * P <0.05, *** P <0.005 and N.S., not significant. Data are representative of at least three independent experiments.

Article Snippet: Human primary keratinocytes were purchased from DS Pharma Biomedical (Tokyo, Japan) and cultured in a serum-free medium for human keratinocytes (DS Pharma Biomedical).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Neutralization, Comparison

Journal: PLoS ONE

Article Title: ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

doi: 10.1371/journal.pone.0105498

Figure Lengend Snippet: ( a ) Immunohistochemical staining of ΔNp63 in normal skin and skin lesions of AD and SLE. Consecutive sections of each condition were stained with hematoxylin and eosin (HE). Bar = 50 µm. ( b ) Level of ΔNp63 was classified into three categories depending on staining intensity, as indicated by ΔNp63 hi , ΔNp63 lo and ΔNp63 − . The dot plot represents ratios of ΔNp63 lo/- keratinocytes in normal skin (n = 3) and AD lesions (n = 10). ( c ) Quantitative RT-PCR demonstrating the levels of TSLP and ΔNp63 mRNAs in normal skin and AD lesions. Student's t test. * P <0.05 and **** P <0.001. Data are representative of at least three independent experiments.

Article Snippet: Human primary keratinocytes were purchased from DS Pharma Biomedical (Tokyo, Japan) and cultured in a serum-free medium for human keratinocytes (DS Pharma Biomedical).

Techniques: Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

doi: 10.1371/journal.pone.0105498

Figure Lengend Snippet: AD lesions possess many ΔNp63 lo/- keratinocytes, which might be considered as a major source of TSLP. A potential TLR3 ligand such as damaged-cell-derived dsRNA suppresses the expression of ΔNp63, which eventually further upregulates TSLPR and IL-7Rα and augments the susceptibility to TSLP in ΔNp63 lo/- keratinocytes. Since TSLP itself can help ΔNp63 lo/- keratinocytes to produce endogenous TSLP under the condition of TLR3 stimulation, an autocrine and/or paracrine loop of TSLP are generated in ΔNp63 lo/- keratinocytes, leading to undesired inflammation of AD lesions.

Article Snippet: Human primary keratinocytes were purchased from DS Pharma Biomedical (Tokyo, Japan) and cultured in a serum-free medium for human keratinocytes (DS Pharma Biomedical).

Techniques: Derivative Assay, Expressing, Generated