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: Scientific Reports

Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

doi: 10.1038/s41598-018-30562-y

Figure Lengend Snippet: Keap1 interacts with MCM3 in mammalian cells. ( a ) Western blots with antibodies against indicated proteins either with nuclear (‘N’) or cytoplasmic (‘C’) extracts of the FLAG-MCM3 expressing CHO-EBNALT85 cells (‘input’), or in MCM3 complexes immunoprecipitated with anti-FLAG affinity beads (‘flag IP’). Histone H3 and GAPDH were used as fractionation controls. See Supplementary Fig. for full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels (top panels) and Western blots with antibodies against indicated proteins (bottom panels) showing distribution of FLAG-MCM3 immunoprecipitated nuclear and cytoplasmic protein complexes in the Superdex 200 size exclusion chromatography. ‘flag’ depicts the lanes with input material. Co-elution of molecular weight markers is indicated at the bottom. See Supplementary Fig. for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p < 0.0005) are derived from unpaired two-tailed t test. ( e ) Immunofluorescence images of the overall distribution of MCM3 and Keap1 proteins in HPEK cells. The images from green protein channel alone are in the left column, and combined with the blue nuclear DAPI staining in the right column. White scale bar = 10 µM.

Article Snippet: Human primary epidermal keratinocyte progenitor cells (single juvenile donor, passage 2) were purchased from CELLnTEC Advanced Cell Systems and grown for no more than 8 passages in CnT-Prime media provided by the supplier.

Techniques: Western Blot, Expressing, Immunoprecipitation, Fractionation, Staining, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Molecular Weight, Ligation, Control, Confocal Microscopy, Negative Control, Standard Deviation, Derivative Assay, Two Tailed Test, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fisetin Promotes Hair Growth by Augmenting TERT Expression

doi: 10.3389/fcell.2020.566617

Figure Lengend Snippet: Screening for polyphenols that activate hTERT transcription in HaCaT cells. (A) Screening for polyphenols that activate hTERT transcription. Polyphenols (10 μM) were added to the HaCaT(hTERTp-EGFP) cells and cultured for 48 h; then, changes in EGFP fluorescence were monitored using an IN Cell Analyzer 1000. (B) The effect of polyphenols on the expression of endogenous hTERT in HaCaT cells was evaluated by qRT-PCR. Statistical significance was determined using a two-sided Student’s t -test. Statistical significance was defined as P < 0.05 (** P < 0.01; *** P < 0.001).

Article Snippet: The HaCaT human keratinocyte cell line (Riken Bioresource Center, Tsukuba, Japan) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, United States) at 37°C in a 5% CO 2 atmosphere.

Techniques: Cell Culture, Fluorescence, Expressing, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fisetin Promotes Hair Growth by Augmenting TERT Expression

doi: 10.3389/fcell.2020.566617

Figure Lengend Snippet: Effects of polyphenols on the expression of cytokine-encoding genes in HaCaT cells. After HaCaT cells were treated with polyphenols, the gene expression levels of IGF-1 (A) , KGF (B) , and TGF- β 1 (C) were measured by qRT-PCR. Statistical significance was determined using a two-sided Student’s t -test. Statistical significance was defined as P < 0.05 (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The HaCaT human keratinocyte cell line (Riken Bioresource Center, Tsukuba, Japan) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, United States) at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fisetin Promotes Hair Growth by Augmenting TERT Expression

doi: 10.3389/fcell.2020.566617

Figure Lengend Snippet: Effects of polyphenols on β-catenin activity. The effect of these polyphenols on the activity of β-catenin was evaluated. (A) The TOP-Flash reporter assay was performed to evaluate β-catenin activity in HaCaT cells treated with polyphenols. (B) The effect of polyphenols on AXIN2 expression was evaluated by qRT-PCR. (C,D) The effect of polyphenols on protein expression of β-catenin was evaluated by western blotting using anti-β-catenin antibody. Band intensities were quantitatively determined using ImageJ software. (E) The effect of polyphenols on activity of β-catenin was evaluated by immunofluorescence study using anti-active β-catenin antibody. Statistical significance was determined using a two-sided Student’s t -test. Statistical significance was defined as P < 0.05 (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The HaCaT human keratinocyte cell line (Riken Bioresource Center, Tsukuba, Japan) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, United States) at 37°C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot, Software, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fisetin Promotes Hair Growth by Augmenting TERT Expression

doi: 10.3389/fcell.2020.566617

Figure Lengend Snippet: Effect of polyphenols on the growth of HaCaT cells. (A) After HaCaT cells were treated with 10 μM of polyphenols, cell proliferation was monitored using the Cell Counting Kit-8. (B) The proliferation index on the 3rd day was determined. Statistical significance was determined using a two-sided Student’s t -test. Statistical significance was defined as P < 0.05 (* P < 0.05).

Article Snippet: The HaCaT human keratinocyte cell line (Riken Bioresource Center, Tsukuba, Japan) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, United States) at 37°C in a 5% CO 2 atmosphere.

Techniques: Cell Counting

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fisetin Promotes Hair Growth by Augmenting TERT Expression

doi: 10.3389/fcell.2020.566617

Figure Lengend Snippet: Role of hTERT in the polyphenol-induced effects on HaCaT cells. (A) Relative expression levels of hTERT in HaCaT cells transduced with retroviruses expressing shRNA targeting hTERT (sh-hTERT1 and 2) and scramble shRNA (SCR) were evaluated by qRT-PCR. HaCaT cells whose hTERT expression was reduced by shRNA (sh-hTERT-1 and 2) were treated with polyphenols, and relative β-catenin activity (B) , cytokine gene expression (C,D) , and cell proliferation (E) was determined. Statistical significance was determined using a two-sided Student’s t -test. Statistical significance was defined as P < 0.05 (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The HaCaT human keratinocyte cell line (Riken Bioresource Center, Tsukuba, Japan) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, United States) at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Transduction, shRNA, Quantitative RT-PCR, Activity Assay, Gene Expression

Journal: bioRxiv

Article Title: RPRM negatively regulates ATM levels involving its phosphorylation mediated by CDK4/CDK6

doi: 10.1101/2021.11.10.468148

Figure Lengend Snippet: a, b Co-immunoprecipitation assay (Co-IP) was performed to demonstrate the interaction between ATM and RPRM, which was enhanced in irradiated H460-RPRM cells. c Representative immunofluorescence images of co-localization of RPRM and ATM in RPRM -/- MEFs reexpressing RPRM at different times post 20 Gy X-irradiation. d, e Change in the levels of total ATM and phospho-ATM in H460-NC (N)/RPRM (R) cells at different times post 2 Gy X-irradiation. Quantification of ATM levels ( e ), which were the average of three independent experiments. f Change in the ATM levels of HaCaT-NC (N)/RPRM (R) cells irradiated with 2 Gy X-rays showed that RPRM overexpression in HaCaT cells decreased ATM levels upon IR. g, h The ATM levels of RPRM +/+ MEFs were lower than those of RPRM -/- MEFs at different times post 20 Gy X-irradiation. Bar graph ( h ) shows quantification of ATM levels. i, j RPRM reexpression reduced the ATM levels in RPRM -/- MEFs post 20 Gy X-irradiation. Bar graph ( j ) shows quantification of ATM levels. Experiments were independently repeated three times with similar results. * P < 0.05; ** P < 0.01.

Article Snippet: HaCaT human immortalized keratinocytes were obtained from China Center for Type Culture Collection.

Techniques: Co-Immunoprecipitation Assay, Irradiation, Immunofluorescence, Over Expression

Journal: PLOS ONE

Article Title: Aromatic oil from lavender as an atopic dermatitis suppressant

doi: 10.1371/journal.pone.0296408

Figure Lengend Snippet: Human keratinocyte-like cell line HaCaT stably expressing the NanoLuc luciferase reporter gene under the control of an XRE (XRE-NLuc::HaCaT) was incubated in culture medium containing 0.5 μM FICZ with or without the indicated amounts of agents for 24 h. The percentage inhibition of AD was calculated by the reporter expression with FICZ alone as 0%, indicating no inhibition, and without FICZ as 100% indicating complete inhibition. The dot markers in the graph represent data from three independent experiments. The vertical and horizontal bars represent the standard deviation and the mean, respectively. a) Comparison of L . angustifolia essential oil and generic lavender oil. b) Comparison of linalyl acetate, linalool, THL, and DHL. The statistical significance of the data points among the different concentrations and the dose-dependency were determined by the Kruskal-Wallis test or one-way ANOVA, and the Williams test, respectively, respectively. *, ** P <0.05 (n = 3).

Article Snippet: Human immortalized keratinocyte cell line HaCaT (#300493, CLI, Cosmo-bio, Tokyo, Japan), established from adult male skin, was maintained at 37°C under 5%CO 2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Stable Transfection, Expressing, Luciferase, Control, Incubation, Inhibition, Standard Deviation, Comparison

Journal: PLOS ONE

Article Title: Aromatic oil from lavender as an atopic dermatitis suppressant

doi: 10.1371/journal.pone.0296408

Figure Lengend Snippet: HaCaT cells were incubated in culture medium containing 5 μM FICZ with or without the indicated amounts of agents for 24 h. Total RNA was then extracted and reverse transcribed to cDNA that was subjected to qPCR using the primer set listed in S2 Table in . Artemin expression was normalized to GAPDH expression. Percentage inhibition was then calculated with FICZ alone as 0%, indicating no inhibition, and without FICZ as 100% indicating complete inhibition. The dot markers in the graphs represent data from two or three independent experiments. The vertical and horizontal bars represent the standard deviation and the mean, respectively. Comparisons of (a) L . angustifolia essential oil and generic lavender oil, and (b) linalyl acetate and linalool for inhibition of induced Artemin expression are shown. The statistical significance of the data points among the different concentrations and the dose-dependency were determined by the Kruskal-Wallis test or one-way ANOVA, and the Williams test, respectively. *, ** P <0.05 (n = 3).

Article Snippet: Human immortalized keratinocyte cell line HaCaT (#300493, CLI, Cosmo-bio, Tokyo, Japan), established from adult male skin, was maintained at 37°C under 5%CO 2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Incubation, Reverse Transcription, Expressing, Inhibition, Standard Deviation

Journal: PLOS ONE

Article Title: Aromatic oil from lavender as an atopic dermatitis suppressant

doi: 10.1371/journal.pone.0296408

Figure Lengend Snippet: a, b) HaCaT cells incubated in culture medium containing the indicated agents for 24 h. Total RNA was then extracted and reverse transcribed to cDNA that was subjected to qPCR using the primer set listed in S2 Table in . AhR and ARNT expression was normalized to GAPDH expression. Fold induction was calculated as the vehicle set at 1. Values in the graphs represent the mean ± SD of two independent experiments. There were no statistically significant differences under all conditions. c, d) HaCaT cells were incubated in culture medium containing the indicated agents for 24 h. c) Cells were lysed and subjected to western blotting with antibodies against AhR, ARNT, and α-tubulin. All the Western blots are shown from two (anti-ARNT) or three (anti-AhR and anti-gamma tubulin) independent experiments. The right-side numbers are experimental numbers. d) Band intensity of each lane was quantified by ImageJ and normalized to α-tubulin. The intensities are relative to the vehicle set at 1. Values in the graphs represent the mean ± SD of two or three independent experiments. * p <0.05, compared with FICZ alone. LA, linalyl acetate.

Article Snippet: Human immortalized keratinocyte cell line HaCaT (#300493, CLI, Cosmo-bio, Tokyo, Japan), established from adult male skin, was maintained at 37°C under 5%CO 2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Incubation, Reverse Transcription, Expressing, Western Blot

Journal: PLOS ONE

Article Title: Aromatic oil from lavender as an atopic dermatitis suppressant

doi: 10.1371/journal.pone.0296408

Figure Lengend Snippet: EC 1.5 and CV 70 in the keratinocyte activation assay.

Article Snippet: Human immortalized keratinocyte cell line HaCaT (#300493, CLI, Cosmo-bio, Tokyo, Japan), established from adult male skin, was maintained at 37°C under 5%CO 2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay

Journal: PLOS ONE

Article Title: Aromatic oil from lavender as an atopic dermatitis suppressant

doi: 10.1371/journal.pone.0296408

Figure Lengend Snippet: Four key events in the skin sensitization reaction and reactivities of L . angustifolia essential oil, generic lavender oil, linalyl acetate, and linalool.

Article Snippet: Human immortalized keratinocyte cell line HaCaT (#300493, CLI, Cosmo-bio, Tokyo, Japan), established from adult male skin, was maintained at 37°C under 5%CO 2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Lymph Node Assay