Journal: BMJ Open Ophthalmology

Article Title: Protective effects of blue light-blocking shades on phototoxicity in human ocular surface cells

doi: 10.1136/bmjophth-2018-000217

Figure Lengend Snippet: Representative fluorescent microscopic images of immunostained cells with anti-pan-keratin antibody and its isotype control antibody (mouse IgG1). (A, B) Cells from human corneal surface origin (peripheral portion) and normal human dermal fibroblasts immunostained with anti-pan-keratin antibody, respectively. (C, D) Cells from human corneal surface origin (peripheral portion) and normal human dermal fibroblasts immunostained with an isotype control antibody (mouse IgG1), respectively. Scale bar=100 µm.

Article Snippet: Normal human dermal fibroblasts (NHDF, DS Pharma Biomedical, Osaka, Japan) were used as a negative control.

Techniques: Control

Journal: International Journal of Cosmetic Science

Article Title: Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing ‐ Experimental evidence for a holistic treatment approach

doi: 10.1111/ics.12784

Figure Lengend Snippet: Antioxidative and anti‐inflammatory capacity of bakuchiol and retinol. (a) Antioxidative efficacy of bakuchiol (100 μM) or retinol (100 μM) compared to the high standard trolox (25 μM) and the solvent control determined by a DPPH antioxidant assay through absorbance measurement at 524 nm after 10, 30 and 60 min. N = 12. (b) Antioxidative power expressed in antioxidative units of bakuchiol, retinol or the high standard vitamin C (vit. C) determined using electron spin resonance spectroscopy. (c) ELISA‐based measurement of prostaglandin E2 (PGE2) levels in unstressed human dermal fibroblasts (HDFs), LPS‐stressed control HDFs, and in LPS‐stressed HDFs treated with the high standard diclofenac (25 ng/mL), bakuchiol or retinol (both applied at 1.25, 2.5, 5, 10 μM) for 24 h. N = 12. (d) Macrophage migration inhibitory factor (MIF) protein levels in unstressed HDFs, in HDFs stressed by a DPBS incubation and in stressed HDFs treated with bakuchiol or retinol (both applied at 1 and 10 μM) for 48 h determined by ELISA. N = 10. Results are depicted as mean ± SD. Statistics were performed by RM‐ANOVA with post‐hoc pairwise comparison based on Blom‐transformed ranks for Figure or by a pairwise Wilcoxon signed rank test for Figure . Significant differences are marked with an asterisk (** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human dermal fibroblasts (HDFs) from multiple donors were obtained from Tissue Solutions Ltd (Glasgow, UK), Lonza (Basel, Switzerland), tebu‐bio (Heerhugowaard, Netherlands) or isolated from full‐thickness skin explants purchased from Alphenyx (Marseille, France) as described before [ ].

Techniques: Solvent, Control, Antioxidant Assay, Electron Paramagnetic Resonance, Spectroscopy, Enzyme-linked Immunosorbent Assay, Migration, Incubation, Comparison, Transformation Assay

Journal: International Journal of Cosmetic Science

Article Title: Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing ‐ Experimental evidence for a holistic treatment approach

doi: 10.1111/ics.12784

Figure Lengend Snippet: Effects of bakuchiol and retinol on cell activity and major ECM components. (a) Fibroblast growth factor 7 (FGF7) protein levels in control HDFs and in HDFs treated with 10 μM bakuchiol or retinol for 24 h. N = 13. (b) Quantification of WST‐1 metabolization in control HDFs and in HDFs treated with 10% triton‐X, bakuchiol or retinol (both applied at 1 and 10 μM) for 72 h. Difference in absorbance at 620 and 450 nm is depicted. N = 12. (c) Collagen, type VII, alpha 1 (COL7A1) protein levels in control HDFs and in HDFs treated with the high standard TGF‐β (10 ng/mL) and sodium ascorbate (SA; 11 μg/mL), as well as with bakuchiol or retinol (4 h: 1 and 10 μM, extended incubation: 10 μM for both test substances) for 4 h or for an extended incubation time (72 h or 96 h depending on cell confluence). N = 10 for 4 h, n = 11 for extended incubation. (d) Collagen, type I, alpha 1 (COL1A1) protein levels in control HDFs and in HDFs treated with the high standard TGF‐β and SA as aforementioned, bakuchiol or retinol (both applied at 1 and 10 μM) for 4 h. N = 11. (e) Fibronectin (FN) protein levels in control HDFs and in HDFs treated with 10 μM bakuchiol or retinol for 24 h. N = 11. All protein levels were determined by ELISA. Results are depicted as mean ± SD. Statistics were performed by RM‐ANOVA with post‐hoc pairwise comparison for Figure or by a pairwise Wilcoxon signed rank test for Figure . Significant differences are marked with an asterisk (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human dermal fibroblasts (HDFs) from multiple donors were obtained from Tissue Solutions Ltd (Glasgow, UK), Lonza (Basel, Switzerland), tebu‐bio (Heerhugowaard, Netherlands) or isolated from full‐thickness skin explants purchased from Alphenyx (Marseille, France) as described before [ ].

Techniques: Activity Assay, Control, Incubation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific Reports

Article Title: The monoclonal antibody EPR1614Y against the stem cell biomarker keratin K15 lacks specificity and reacts with other keratins

doi: 10.1038/s41598-018-38163-5

Figure Lengend Snippet: Organotypic cultures (OTCs) of keratinocyte cell lines. HaCaT ( A ), N/Tert-1 ( B ), KEB-11 ( C ) and NEB-1 ( D ) cells were co-cultured with primary dermal fibroblasts in OTCs for 7 and 9 days, followed by paraffin embedding and sectioning. Images of H&E-stained specimens were acquired using Leica Epi DM5000B or Nikon Eclipse 80i microscopes; scale bar = 200 μm.

Article Snippet: HaCaT, N/Tert-1, KEB-11 and NEB-1 keratinocytes were seeded on a human primary dermal fibroblast containing disc of rat-tail collagen type I (Corning, UK) in a Millipore ® Millicell ® (Sigma-Aldrich, UK).

Techniques: Cell Culture, Staining

Journal: Gene Therapy

Article Title: Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity

doi: 10.1038/gt.2014.11

Figure Lengend Snippet: Transgene integration at 8p22 and durable FVIII secretion in phiC31 integrase-modified primary adult human cells. ( a ) G418-resistant stable cells derived from primary cultures of bone marrow-derived stromal cells (BMSC), adipose-derived stromal cells (ADSC) and normal dermal fibroblasts (NF123) co-electroporated with p attB hybrid FVIII and phiC31 integrase were examined for evidence of transgene integration into intron 7 of DLC1 by left and right integration junction PCR. ‘–ve' refers to minus template amplification. Left and right integration junctions were amplified with vector-specific and genomic DNA-specific primers while control genomic PCR was performed with a pair of genome specific-primers amplifying a 900-bp region within chromosome 19. Amplified products were electrophoresed on 1% agarose gels and imaged using BioRad Gel Doc 2000 transilluminator and QuantityOne software. A composite image of the original gel is shown. Black arrows indicate the predicted integration junction PCR amplified bands. ( b ) FVIII activity in overnight conditioned media of untreated (Wt) and G418-resistant (phiC31 integrase-modified) BMSC, ADSC and NF123, determined 1 month after co-electroporation with p attB hybrid FVIII and phiC31 integrase. Data are mean±s.e.m.; n =3 per group.

Article Snippet: Primary human dermal fibroblasts (KF1 and NF123), human adipose-derived stromal cells, human bone marrow-derived stromal cells and CLECs were provided by CellResearch Corporation, Singapore.

Techniques: Modification, Derivative Assay, Amplification, Plasmid Preparation, Control, Software, Activity Assay, Electroporation

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Platelet polyphosphate induces fibroblast chemotaxis and myofibroblast differentiation

doi: 10.1111/jth.15066

Figure Lengend Snippet: (A) NIH-3T3 cells or (B) GM1381 cells were incubated in 1.5% serum in the presence or absence of releasates from activated human platelets (diluted to 15 μg/mL protein). As indicated, some releasates were previously heat-treated to denature proteins, or digested with ScPPX1 to destroy polyphosphate (red indicates treatment with ScPPX1). After 48 hours, antibody staining for α-SMA was quantified by confocal microscopy as a marker of myofibroblast differentiation. All values are mean ± SEM (n = 5). Asterisks highlight statistical significance of the indicated comparisons: * p < 0.05, ** p <0.01, *** p <0.001 (paired t-tests).

Article Snippet: Cell culture The murine embryonic fibroblastic cell line, NIH-3T3 (ATCC CRL-1658), and primary human dermal fibroblasts, GM1381 (Coriell Institute), were routinely cultured in DMEM supplemented with 10 mmol/L HEPES, penicillin, streptomycin, 4 mmol/L L-glutamine, and 10% bovine calf serum, in a 5% CO 2 environment at 37°C.

Techniques: Incubation, Staining, Confocal Microscopy, Marker

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Platelet polyphosphate induces fibroblast chemotaxis and myofibroblast differentiation

doi: 10.1111/jth.15066

Figure Lengend Snippet: (A-C) Platelet-size polyphosphate (polyP) increases α-SMA levels in NIH-3T3 cells. (A) Representative images in which NIH-3T3 cells were cultured in 1.5% serum in the absence (control) or presence of 5 μmol/L platelet-sized polyphosphate. After 48 hours, cells were stained for α-SMA (red) or DNA (green) and imaged by confocal microscopy (bar = 10 μm). (B,C) Dose-response of NIH-3T3 cells to platelet-size polyphosphate. In 5 experiments, cells were treated for 48 hours in 1.5% serum plus the indicated concentrations of platelet-size polyphosphate, after which the following were quantified: (B) integrated intensity of α-SMA staining per cell, or (C) percent cells positive for α-SMA staining localized along fibrin tendrils (stress fibers). (D) Platelet-size polyphosphate increases cell body area. NIH-3T3 cells were cultured in 1.5% serum in the absence or presence of 1 μmol/L polyphosphate for 48 hours and imaged by confocal microscopy. Cell body area of individual cells was quantified. (E-F) Polymer length-dependence of polyphosphate’s ability to induce myofibroblast differentiation in NIH 3T3 cells. Cells were incubated in 1.5% serum for 48 hours in the presence or absence of 1 μmol/L polyphosphate of the indicated polymer lengths (in phosphate units): (E) integrated intensity of α-SMA staining per cell, or (F) percent cells positive for α-SMA staining localized along fibrin tendrils (stress fibers). (G) Dose-response of GM1381 cells to platelet-size polyphosphate. Cells were incubated in 1.5% serum with varying concentrations of platelet-size polyphosphate for 48 hours, after which the integrated intensity of α-SMA staining was quantified. Values in panels B-C and E-G are mean ± SEM (n = 5). Asterisks highlight statistical significance relative to the no-polyphosphate controls: * p < 0.05, ** p <0.01, *** p <0.001 (paired t-tests). Values in panel D are mean ± SEM of individual cells across five experiments. **** p <0.0001 (unpaired t-test).

Article Snippet: Cell culture The murine embryonic fibroblastic cell line, NIH-3T3 (ATCC CRL-1658), and primary human dermal fibroblasts, GM1381 (Coriell Institute), were routinely cultured in DMEM supplemented with 10 mmol/L HEPES, penicillin, streptomycin, 4 mmol/L L-glutamine, and 10% bovine calf serum, in a 5% CO 2 environment at 37°C.

Techniques: Cell Culture, Control, Staining, Confocal Microscopy, Polymer, Incubation