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: International Journal of Molecular Sciences

Article Title: Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway

doi: 10.3390/ijms18091948

Figure Lengend Snippet: ( A ) Localization of NRF2 is visualized by an immunofluorescence technique. NRF2 is mainly located in the cytoplasm of control keratinocytes. Nuclear staining of NRF2 is enhanced in the keratinocytes treated with APE for 6 h. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 20 μm; ( B ) the number of NHEKs with nuclear-predominant staining of NRF2 is significantly increased by APE treatment than that of control; ( C ) APE upregulates the gene expression of antioxidant enzyme NQO1 ; and ( D ) APE also upregulates the gene expression of antioxidant enzyme HO1 . ** p < 0.01.

Article Snippet: The NHEKs were cultured in normal human epidermal keratinocyte proliferation medium, HuMedia-KG2 (KG2; Kurabo, Osaka, Japan), containing insulin, human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, and antibacterial substances.

Techniques: Immunofluorescence, Control, Staining, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway

doi: 10.3390/ijms18091948

Figure Lengend Snippet: ( A ) Localization of AHR is visualized by an immunofluorescence technique. AHR is mainly located in the cytoplasm of control keratinocytes. Nuclear staining of AHR is enhanced in the keratinocytes treated with APE for 6 h. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 20 μm; ( B ) the number of NHEKs with nuclear-predominant staining of AHR is significantly increased by APE treatment than that of control; ( C ) APE dose-dependently upregulates the gene expression of CYP1A1 , a specific AHR-responsive metabolizing enzyme. ** p < 0.01; and ( D ) APE-induced CYP1A1 upregulation is canceled in keratinocytes transfected with AHR siRNA. ** p < 0.01.

Article Snippet: The NHEKs were cultured in normal human epidermal keratinocyte proliferation medium, HuMedia-KG2 (KG2; Kurabo, Osaka, Japan), containing insulin, human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, and antibacterial substances.

Techniques: Immunofluorescence, Control, Staining, Gene Expression, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway

doi: 10.3390/ijms18091948

Figure Lengend Snippet: APE (0.03%)-induced FLG ( A ) and LOR ( B ) upregulation is canceled in keratinocytes with AHR knockdown. APE-induced FLG ( C ) and LOR ( D ) upregulation is also inhibited in keratinocytes with OVOL1 knockdown. * p < 0.05.

Article Snippet: The NHEKs were cultured in normal human epidermal keratinocyte proliferation medium, HuMedia-KG2 (KG2; Kurabo, Osaka, Japan), containing insulin, human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, and antibacterial substances.

Techniques: Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Adjustments of the Phytochemical Profile of Broccoli to Low and High Growing Temperatures: Implications for the Bioactivity of Its Extracts

doi: 10.3390/ijms25073677

Figure Lengend Snippet: The impact of different concentrations of extracts from broccoli cultivated under room (RT), high (HT), and low (LT) temperature on the levels of ROS within ( a ) mouse embryonal fibroblasts (MEF), ( b ) normal human keratinocytes (HaCaT), ( c ) hepatocellular carcinoma (HepG2), ( d ) colorectal carcinoma (HCT116), and ( e ) lung carcinoma (H460) cell cultures. Values represent mean ± standard deviation of three biological replicates. Different letters indicate a significant difference between the RT, HT, and LT broccoli microgreens, separately for each extract concentration (ANOVA, Duncan test, p ≤ 0.05). An asterisk (*) indicates a significant difference between each group of cells treated with broccoli microgreen extracts and control cells (Student’s t -test, p ≤ 0.05). A double asterisk (**) indicates a significant difference between cells treated with H 2 O 2 only and cells treated simultaneously with H 2 O 2 and broccoli extract at a 0.05 mg/mL concentration (Student’s t -test, p ≤ 0.05). AU = arbitrary units; Con = control cells; DCF = dichlorodihydrofluorescein; FI = fluorescence intensity.

Article Snippet: Intracellular ROS levels were determined on mouse embryonal fibroblasts (MEF, established in the laboratory of Dr. Pinteric from mice RRID:IMSR_JAX:002448, Jackson Lab, Bar Harbor, ME, USA), normal human keratinocytes (HaCaT, RRID: CVCL-0038, a gift from Dr. Marijeta Kralj), hepatocellular carcinoma (HepG2, ATCC, HB-8065, Manassas, VA USA), colorectal carcinoma (HCT116, ATCC, CCL-247, Manassas, VA, USA), and lung carcinoma (H460, ATCC, HTB-177, Manassas, VA, USA) cell lines using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, cat. no. D6883, Sigma Aldrich, Saint Louis, MO, USA).

Techniques: Standard Deviation, Concentration Assay, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Adjustments of the Phytochemical Profile of Broccoli to Low and High Growing Temperatures: Implications for the Bioactivity of Its Extracts

doi: 10.3390/ijms25073677

Figure Lengend Snippet: The principal component analysis showing ( a ) the relation between broccoli microgreens grown under room (RT), high (HT), and low (LT) temperatures based on the analyzed variables, whose grouping is shown in the ( b ) part of the figure. ABA = abscisic acid; Car = carotenoids; cc = conjugated compound; Chl = chlorophyll; fc = free compound; FerA= ferulic acid; GLS = total intact glucosinolates; H2O2 = hydrogen peroxide; H460 = lung carcinoma; HaCaT = normal human keratinocytes; HCT116 = colorectal carcinoma; HepG2 = hepatocellular carcinoma; IAA = indole-3-acetic acid; K = kaempferol; L-asc = L -ascorbic acid, MEF = mouse embryonal fibroblasts; Porf = porphyrins; PROT = total proteins; Q = quercetin; SinA = sinapic acid; SS = total soluble sugars; TA = total monomeric anthocyanins; TF = total flavonoids; TFlo = total flavonols; THCA = total hydroxycinnamic acids; TP = total phenolics; TPA = total phenolic acids; TPAN = total proanthocyanidins; TT = total tannins.

Article Snippet: Intracellular ROS levels were determined on mouse embryonal fibroblasts (MEF, established in the laboratory of Dr. Pinteric from mice RRID:IMSR_JAX:002448, Jackson Lab, Bar Harbor, ME, USA), normal human keratinocytes (HaCaT, RRID: CVCL-0038, a gift from Dr. Marijeta Kralj), hepatocellular carcinoma (HepG2, ATCC, HB-8065, Manassas, VA USA), colorectal carcinoma (HCT116, ATCC, CCL-247, Manassas, VA, USA), and lung carcinoma (H460, ATCC, HTB-177, Manassas, VA, USA) cell lines using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, cat. no. D6883, Sigma Aldrich, Saint Louis, MO, USA).

Techniques:

Journal: Scientific Reports

Article Title: Assessment of cytotoxicity and antioxidant properties of berry leaves as by-products with potential application in cosmetic and pharmaceutical products

doi: 10.1038/s41598-021-82207-2

Figure Lengend Snippet: Cytotoxicity assessment of water-glycerin extracts from leaves of Ribes nigrum L., Vaccinium myrtillus L, Rubus fruticosus L. and Fragaria vesca L . on fibroblasts (BJ) ( A , B ) and keratinocytes (HaCaT) cells ( C , D ) after 24-h exposure assessed by the Alamar Blue assay ( A , C ) and Neutral Red assay ( B , D ). Data are the mean ± SD of three independent experiments in which each concentration tested was performed in four replications. The control was cells not treated with extracts, whose viability was assumed to be 100%. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.03 versus the control (100%).

Article Snippet: HaCaT cells (normal human keratinocytes) were purchased from CLS Cell Lines Service (Germany).

Techniques: Alamar Blue Assay, Neutral Red Assay, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Assessment of cytotoxicity and antioxidant properties of berry leaves as by-products with potential application in cosmetic and pharmaceutical products

doi: 10.1038/s41598-021-82207-2

Figure Lengend Snippet: The effect of water-glycerin leaf extracts from Ribes nigrum L., Vaccinium myrtillus L, Rubus fruticosus L. and Fragaria vesca L. (at a concentration of 0.5–2.5%) on the migration of HaCaT cells after a 24-h incubation in a scratch test. Photographs were taken under a magnification of × 10 under a microscope. The analyzes were performed in triplicate from three independent experiments.

Article Snippet: HaCaT cells (normal human keratinocytes) were purchased from CLS Cell Lines Service (Germany).

Techniques: Concentration Assay, Migration, Incubation, Microscopy

Journal: Nature Communications

Article Title: Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways

doi: 10.1038/s41467-020-20108-0

Figure Lengend Snippet: Effects of ManN on non-endothelial cells of bovine, mouse, or human origin. ManN did not promote growth of Calu6 ( a ), A673 ( b ), U87MG ( c ), and 4T1 ( d ) tumor cells. Ten-percent FBS was used as positive control for Calu6 and A673, whereas 10 ng/ml bFGF and 1 μg/ml human apo-transferrin were used as positive controls for U87MG and 4T1, respectively. Similarly, no increases in proliferation were induced by ManN on AML12 ( e ), bovine pituitary cells ( f ), NIH3T3 cells ( g ), human RPEs ( h ), human dermal fibroblasts ( i ), and human keratinocytes ( j ), alone or in combination with growth factors. Proliferation quantification was performed using AlamarBlue® or MTS (for 4T1 cells). n = 3 independent samples. Inserted are representative western blot analyses showing dose-dependent effects of ManN and mannose at 400 μM (2,4) and 2 mM (3,5) on bFGFR1 or β1 integrin (for 4T1, AML12, NIH3T3 cells, human skeletal muscle cells, human dermal fibroblasts, and human keratinocytes) molecular mass compared to the untreated control (1). β-actin served as loading control. GM: growth media. Proteins were separated on NuPAGE 3–8% Tris-Acetate gel for western blot analysis. For each study, a representative experiment is shown from two independent studies. Asterisks indicate a significant difference compared with control. When statistical analysis was done using a different control, bracket was used between specified groups. Data were means +/− SD of the mean or an average when n = 2. Statistical analysis was done by two-tailed, two-sample unequal variance t test. * p < 0.05, ** p < 0.01. Data are provided as a Source data file.

Article Snippet: Keratinocytes (ATCC, PCS-200-011) were cultured in dermal cell basal media (PCS-200-030) plus keratinocyte growth kit (PCS-200-040).

Techniques: Positive Control, Western Blot, Control, Two Tailed Test