Journal: International Journal of Molecular Sciences

Article Title: Jellyfish Polysaccharides for Wound Healing Applications

doi: 10.3390/ijms231911491

Figure Lengend Snippet: Scratch test on HaCat human keratinocytes cell monolayers treated with RP-JSP1 and RP-JSP2. Control consists in untreated cells. ( a ) Representative micrographs (4× magnification) of treated and control monolayers; ( b ) confluency rate calculated as percentage of scratch closure in respect to the initial scratch area.

Article Snippet: Fibroblast BALB/3T3 clone A31 (CCL-163) and keratinocyte HaCat cell line were purchased from American Type Colture Collection (USA) and CellSystems (GmbH Germany), respectively.

Techniques: Control

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: International Journal of Medical Sciences

Article Title: Gentisic Acid Stimulates Keratinocyte Proliferation through ERK1/2 Phosphorylation

doi: 10.7150/ijms.36484

Figure Lengend Snippet: Gentisic acid induced skin cells proliferation. HaCaT (A) and CCD-986sk (B) cells were treated with various concentrations of gentisic acid. The cell proliferation rate was evaluated with an MTT assay. (** : P < 0.01, *** : P < 0.001)

Article Snippet: The human keratinocyte cell line HaCaT (HaCaT cell was kindly provided by COSMAX BIO, Jecheon, Korea) and human skin fibroblast cell line CCD-986sk (CCD-986sk was purchased from the Korean cell line, Seoul, Korea) was cultured in Dulbecco's Modified Eagle's Medium containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin at 37°C in a 5% CO 2 atmosphere .

Techniques: MTT Assay

Journal: International Journal of Medical Sciences

Article Title: Gentisic Acid Stimulates Keratinocyte Proliferation through ERK1/2 Phosphorylation

doi: 10.7150/ijms.36484

Figure Lengend Snippet: Gentisic acid increased in vitro wound healing. HaCaT cells were cultured in a 6-well plate, scratched, and treated with different concentrations of DMSO only (negative control), gentisic acid, or allantoin (positive control). The results were imaged to show scratched wound healing with various compound concentrations.

Article Snippet: The human keratinocyte cell line HaCaT (HaCaT cell was kindly provided by COSMAX BIO, Jecheon, Korea) and human skin fibroblast cell line CCD-986sk (CCD-986sk was purchased from the Korean cell line, Seoul, Korea) was cultured in Dulbecco's Modified Eagle's Medium containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin at 37°C in a 5% CO 2 atmosphere .

Techniques: In Vitro, Cell Culture, Negative Control, Positive Control

Journal: International Journal of Medical Sciences

Article Title: Gentisic Acid Stimulates Keratinocyte Proliferation through ERK1/2 Phosphorylation

doi: 10.7150/ijms.36484

Figure Lengend Snippet: Gentisic acid induced ERK1/2 phosphorylation in HaCaT cells. HaCaT cells were treated with various concentration of gentisic acid and the protein extracts were used in Western blot analysis. (A) GAPDH was used as a control. p38, ERK1/2, and JNK, which in their phosphorylated forms are associated with cell proliferation, were detected. (B) p-ERK1/2 was quantified with densitometric analysis and normalized to GAPDH. (* : P < 0.05)

Article Snippet: The human keratinocyte cell line HaCaT (HaCaT cell was kindly provided by COSMAX BIO, Jecheon, Korea) and human skin fibroblast cell line CCD-986sk (CCD-986sk was purchased from the Korean cell line, Seoul, Korea) was cultured in Dulbecco's Modified Eagle's Medium containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin at 37°C in a 5% CO 2 atmosphere .

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Control

Journal: EMBO Molecular Medicine

Article Title: A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea

doi: 10.15252/emmm.202013560

Figure Lengend Snippet: The mRNA expression level of human cathelicidin ( CAMP ) in skin lesions from healthy individuals ( n = 10) and patients with rosacea ( n = 15). Co‐immunostaining of cathelicidin and pS6 on skin sections from HS and rosacea. Scale bar, 50 μm. Epi, epidermis. Immunostaining of mouse cathelicidin (CRAMP) in skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle ( n = 6 for each group). Bottom right panels, magnified images of dotted line areas. Epi, epidermis; Der, dermis; Scale bar: 50 μm. Quantification of relative fluorescence intensity for CRAMP in epidermis ( n = 6). Immunoblotting of cathelicidin in cell lysates from primary human keratinocytes treated with different doses of LL37 (0–8 μM) and scramble LL37 (8 μM) for 24 h. Cathelicidin protein levels were analyzed. Immunoblot analysis of cathelicidin and pS6 in cell lysates from primary human keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. Cathelicidin protein levels were analyzed. Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least three independent experiments. Data represent the mean ± SEM. ** P < 0.01. Two‐tailed unpaired Student's t ‐test (A) or 1‐way ANOVA with Bonferroni's post hoc test (D) was used.

Article Snippet: HaCaT keratinocyte cell line obtained from NTCC (Biovector Science Lab, Beijing, China) was cultured in DMEM supplemented with 10% fetal bovine serum, penicillin–streptomycin, and 2 mM glutamine (Invitrogen).

Techniques: Expressing, Immunostaining, Injection, Control, Fluorescence, Western Blot, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea

doi: 10.15252/emmm.202013560

Figure Lengend Snippet: A Representative images showing cellular localization of LL37 or sLL37 analyzed by fluorescent microscope in live primary human keratinocytes treated with FITC‐labeled LL37 or sLL37 (4 μM) for 30 min followed by PBS wash. BF, bright field. Scale bar: 50 μm. B Representative images showing membrane localization of LL37 or sLL37 in live primary human keratinocytes treated with FITC‐labeled LL37 / sLL37 and PKH26 for 30 min. Scale bar: 50 μm. C Immunoblotting analysis of TLR2 and pS6 in cell lysates from HaCaT keratinocytes carrying either a vector expressing a scramble shRNA or TLR2 shRNAs was exposed to LL37 (4 μM) for 2 h. pS6 protein levels were analyzed relative to total S6 (fold change). D Immunoblot analysis of TLR2 and pS6 in cell lysates from HaCaT keratinocytes carrying either a vector expressing TLR2 or an empty vector was exposed to LL37 (4 μM) for 2 h. pS6 protein levels were analyzed relative to total S6. Tubulin or actin was used as the loading control. E Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC‐labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS wash, and then, live cells were analyzed by fluorescent microscope. Scale bar: 50 μm. F, G Primary human keratinocytes were incubated with sLL37‐flag or LL37‐flag for 1 h. Cell lysates were immunoprecipitated with anti‐flag or anti‐TLR2 antibodies, showing an interaction between LL37 and TLR2. Medium, the medium of primary human keratinocytes treated with sLL37‐flag or LL37‐flag. All results are representative of at least three independent experiments.

Article Snippet: HaCaT keratinocyte cell line obtained from NTCC (Biovector Science Lab, Beijing, China) was cultured in DMEM supplemented with 10% fetal bovine serum, penicillin–streptomycin, and 2 mM glutamine (Invitrogen).

Techniques: Microscopy, Labeling, Membrane, Western Blot, Plasmid Preparation, Expressing, shRNA, Control, Incubation, Immunoprecipitation