Journal: Biology Direct

Article Title: BRD4 sustains p63 transcriptional program in keratinocytes

doi: 10.1186/s13062-024-00547-1

Figure Lengend Snippet: BRD4 and p63 regulates keratinocytes proliferation and differentiation. a, b, e, f Cell cycle analysis performed in HEKn comparing SCR condition and sip63 and siBRD4 conditions comparing untreated cells (DMSO) and JQ1 treated cells (10 µM). Cells were stained with Propidium Iodide (50 µg/ml) for 1 h and then analysed by flow cytometry. In a, e representative plot of cell cycle analysis performed in HEKn is shown, while in b, f a quantification of cell cycle analysis. Graphs present means ± SD of three independent experiments. c, g EdU-incorporation assay of HEKn cells transfected with siSCR, sip63 and different BRD4 (siBRD4#1, BRD4#2) siRNAs or treated with DMSO and JQ1 (10 µM). Data are shown as mean ± SD of N = 3 experiments. (Unpaired Student’s t test). d, h Growth curve of HEKn cells transfected with siSCR, sip63 and siBRD4#1, or treated with DMSO and JQ1 (10 µM). The cell confluency has been determined using the Incucyte real-time video imaging system. Each data points indicate mean ± SEM. i – n Western blot analysis was carried out with specific antibodies against K10, and β-actin was used as loading control. ImageJ program was used to quantitate the protein levels. The blot is representative of three independent experiments. DD, days of differentiation

Article Snippet: Primary Normal Human Epidermal Keratinocytes, neonatal (HEKn) (Gibco, catalog no. C-001-5C) and human TERT-immortalized keratinocytes (Ker-CT) (ATCC, CRL4048, lot. no. 0213) were cultured in EpiLife medium with the addition of Human Keratinocyte Growth Supplements (HKGS, Life Technologies).

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Transfection, Imaging, Western Blot, Control

Journal: Biology Direct

Article Title: BRD4 sustains p63 transcriptional program in keratinocytes

doi: 10.1186/s13062-024-00547-1

Figure Lengend Snippet: Keratinocytes lacking for p63 and BRD4 have similar expression profiles. Gene expression analysis performed using nanoString technology on HEKn cells after p63 and BRD4 silencing and JQ1 treatment. a Venn diagram represents common dysregulated genes in the different conditions. b – d Pathway gene ontology of common dysregulated genes. e – g Expression levels of the shared dysregulated genes from the top five enriched categories of the GO-pathway analysis

Article Snippet: Primary Normal Human Epidermal Keratinocytes, neonatal (HEKn) (Gibco, catalog no. C-001-5C) and human TERT-immortalized keratinocytes (Ker-CT) (ATCC, CRL4048, lot. no. 0213) were cultured in EpiLife medium with the addition of Human Keratinocyte Growth Supplements (HKGS, Life Technologies).

Techniques: Expressing, Gene Expression

Journal: Archives of Toxicology

Article Title: Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro

doi: 10.1007/s00204-023-03516-1

Figure Lengend Snippet: Effect of ABT-199 on cell viability of SCL-1 and NHEK. A Chemical structure of ABT-199/venetoclax. To determine the effect of ABT-199 on cell viability, SCL-1 carcinoma cells (B) and keratinocytes (NHEK) (C) were treated with different concentrations of ABT-199 for 96 h. Cell viability was measured by MTT assay. Mock-treated control was set at 100%. Data represent means ± SEM of at least three independent experiments ( n ≥ 3). One-way ANOVA with Dunnett’s multiple comparison test was used for the determination of statistical significance compared to mock-treated control; ** p < 0.01, *** p < 0.001

Article Snippet: For treatment, SCL-1 and A375 were incubated in high glucose (4500 mg/L) DMEM without FBS, while NHEK were grown in keratinocyte basal medium (C-20211, PromoCell).

Techniques: MTT Assay, Control, Comparison

Journal: Archives of Toxicology

Article Title: Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro

doi: 10.1007/s00204-023-03516-1

Figure Lengend Snippet: Selective effect of GP on cell viability of skin cells. A Chemical structure of gossypol (GP). To examine the effect of GP on cell viability, SCL-1 carcinoma cells and keratinocytes (NHEK) were treated with different concentrations of GP for 96 h or mock treated (0) and cell viability was measured by MTT (B) and SRB (C) assay. Mock-treated controls were set at 100%. Data represent means ± SEM of at least three independent experiments ( n ≥ 3). Student’s t test was used for the determination of statistical significance; ** p < 0.01, *** p < 0.001. D, E IC 50 values were calculated by non-linear curve fit analysis (GraphPad Prism 5) based on MTT (D) and SRB assay (E)

Article Snippet: For treatment, SCL-1 and A375 were incubated in high glucose (4500 mg/L) DMEM without FBS, while NHEK were grown in keratinocyte basal medium (C-20211, PromoCell).

Techniques: Sulforhodamine B Assay

Journal: Archives of Toxicology

Article Title: Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro

doi: 10.1007/s00204-023-03516-1

Figure Lengend Snippet: Intracellular amount of GP in SCL-1 cells and keratinocytes (NHEK). Cells were harvested after treatment with 5 µM GP for 0.25, 1, and 2 h and analyzed by HPLC. The amount of intracellular GP was related to the respective protein level. Experiments were performed in triplicate ( n = 3)

Article Snippet: For treatment, SCL-1 and A375 were incubated in high glucose (4500 mg/L) DMEM without FBS, while NHEK were grown in keratinocyte basal medium (C-20211, PromoCell).

Techniques:

Journal: Archives of Toxicology

Article Title: Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro

doi: 10.1007/s00204-023-03516-1

Figure Lengend Snippet: Mitochondrial respiration of GP-treated normal keratinocytes (NHEK). A After treatment with different concentrations of GP or mock treated for 1 h, the oxygen consumption rate (OCR) was measured after successive injection of oligomycin (Oligo), FCCP, and rotenone/antiymcin A (Rot/AA) by Seahorse XF Analyzer. Representative curves are depicted. B–D Based on the OCR in response to these mitochondrial stressors, ATP production (B) , spare respiratory capacity (C), and proton leak (D) were calculated. The values of untreated cells were set at 100%. Data represent means ± SEM of three independent experiments ( n = 3). One-way ANOVA with Dunnett’s multiple comparison test was used for the determination of statistical significance between mock treated and GP-treated cells; * p < 0.01

Article Snippet: For treatment, SCL-1 and A375 were incubated in high glucose (4500 mg/L) DMEM without FBS, while NHEK were grown in keratinocyte basal medium (C-20211, PromoCell).

Techniques: Injection, Comparison

Journal: Cells

Article Title: Effect of SUV39H1 Histone Methyltransferase Knockout on Expression of Differentiation-Associated Genes in HaCaT Keratinocytes

doi: 10.3390/cells9122628

Figure Lengend Snippet: Expression of LCE1 genes. ( A ) Upregulation of LCE1A-F transcripts in differentiated HaCaT cells (left panel) and primary human keratinocytes, NHEK (right panel), in comparison to undifferentiated cells (control). ( B ) Fold difference in expression of LCE1A , LCE1B , and LCE1C in undifferentiated (left panel) and differentiated (right panel) SUV39H1-KO HaCaT cells over WT HaCaT cells. The bars represent mean ± SEM calculated from the results of n = 3–5 (depending on the gene) RT-qPCR experiments. Each experiment consisted of three technical replicates and was performed on a different batch of RNA. Statistically significant differences are indicated by horizontal bars and the respective p -values.

Article Snippet: Primary human keratinocytes, NHEK (PromoCell, Heidelberg, Germany), were cultured in Keratinocyte Growth Medium 2 (PromoCell, Heidelberg, Germany) with growth supplement as described in [ ].

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of biomedical science

Article Title: Hesperetin activates CISD2 to attenuate senescence in human keratinocytes from an older person and rejuvenates naturally aged skin in mice.

doi: 10.1186/s12929-024-01005-w

Figure Lengend Snippet: Fig. 1 CISD2 expression is down-regulated in sun-exposed sites of the human skin epidermis. A Fluorescent immunohistochemistry (IHC) staining of CISD2 and KRT14 (a marker of the proliferating keratinocytes in the epidermis) in normal human skin. B The icon shows the various collection sites on normal human skin that are present in the human skin tissue array (SKN1001). The sun-exposed sites are marked with an asterisk. C Quantification of CISD2 intensity in the various different sites of human normal skin. D Quantification of KRT14 intensity in various different sites of human normal skin. E Correlation analysis between CISD2 and KRT14 intensity. F CISD2 mRNA expression in the human skin from the suprapubic area (non-sun-exposed) and the lower leg area (sun-exposed). These data were collected from the Human Protein Atlas database (https://www. proteinatlas.org). Data are presented as mean ± SD. *p < 0.05; **p < 0.005. The statistical analysis was performed by Student’s t test

Article Snippet: The HEK001 (CRL-2404, ATCC) human keratinocyte cell line, which was obtained from an older human subject, was cultured in keratinocyte-serum free medium (Gibco, Carlsbad, CA, USA, 17005-042) supplemented with human recombinant EGF (5 ng/mL), gentamicin (10 mg/mL), and 2 mM L-glutamine.

Techniques: Expressing, Immunohistochemistry, Marker

Journal: Journal of biomedical science

Article Title: Hesperetin activates CISD2 to attenuate senescence in human keratinocytes from an older person and rejuvenates naturally aged skin in mice.

doi: 10.1186/s12929-024-01005-w

Figure Lengend Snippet: Fig. 2 Hesperetin enhances CISD2, promotes mitochondrial function, alleviates oxidative stress and UVB-induced damage in HEK001 keratinocytes. A Real-time RT-qPCR of CISD2 mRNA in Ker-CT and HEK001 keratinocytes. CISD2 levels were normalized to HPRT1. B Western blot analysis of CISD2 protein in the vehicle (Veh)- and hesperetin (Hes)-treated HEK001 keratinocytes. C The mitochondrial oxygen consumption rates (OCR) of HEK001 keratinocytes after different treatments (n = 10–12). OA, Oligomycin A; Rot/AA, rotenone/antimycin A. D Western blot analysis of CISD2 protein in the shLuc and CISD2 knockdown (KD) HEK001 keratinocytes. E The mitochondrial OCR of HEK001 keratinocytes after different treatments (n = 8). F Intracellular H2O2 levels. To assess H2O2-induced oxidative stress, HEK001 keratinocytes were treated with 5 μM H2O2 for 5 min before monitoring the intracellular H2O2 levels. G Mitochondrial membrane potential and quantification of the red/green ratios by JC-1 staining of HEK001 keratinocytes. For hesperetin treatment, 10 μM hesperetin was used to treat the shLuc and CISD2 KD HEK001 keratinocytes for 48 h and mitochondrial membrane potential was monitored before and after 98 μM H2O2 treatment. H Protocol for the treatment with hesperetin and its effect on UVB-induced MMP-1 activation in HEK001 keratinocytes. I Western blot analysis of MMP-1 protein in the Veh- and Hes-treated HEK001 keratinocytes after UVB exposure. J ELISA analysis of pro-MMP-1 protein in the conditioned medium from various groups of HEK001 keratinocytes. Vehicle, 0.1% DMSO. All the experiments were performed and repeated three independent times as biological replicates using HEK001 keratinocytes. Data are presented as mean ± SD. In (C and J), the statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test. In (E and F), the statistical analysis was performed by two-way ANOVA with Bonferroni multiple comparison test. In (A), (B) and (G), the statistical analysis was performed using Student’s t test; not significant (n.s.)

Article Snippet: The HEK001 (CRL-2404, ATCC) human keratinocyte cell line, which was obtained from an older human subject, was cultured in keratinocyte-serum free medium (Gibco, Carlsbad, CA, USA, 17005-042) supplemented with human recombinant EGF (5 ng/mL), gentamicin (10 mg/mL), and 2 mM L-glutamine.

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Membrane, Staining, Activation Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of biomedical science

Article Title: Hesperetin activates CISD2 to attenuate senescence in human keratinocytes from an older person and rejuvenates naturally aged skin in mice.

doi: 10.1186/s12929-024-01005-w

Figure Lengend Snippet: Fig. 5 Hesperetin modulates a panel of DEGs to attenuate senescence and maintain cellular homeostasis. A CISD2 and KRT4 mRNA levels are up-regulated in the HEK001 keratinocytes after hesperetin (Hes) treatment for 48 h. Vehicle (Veh), 0.1% DMSO. B Principal component analysis (PCA, EZinfo 3.0.3 software) of all transcriptomic data (12,103 genes) from vehicle or hesperetin treated HEK001 keratinocytes (n = 3 independent trials). C Volcano plot revealing transcriptome change (Hes vs. Veh). Horizontal line shows the 0.5% false discovery rate (FDR) threshold. Red or blue plots identify genes above the indicated FDR and fold change threshold. A total of 1723 differentially expressed genes (DEGs) were found to be modulated by hesperetin (821 up-regulated and 902 down-regulated genes; Hes vs Veh, Fold change > 1.5 and FDR < 0.005). D The biological processes obtain from Gene Ontology (GO) annotation of transcriptome changes (1723 DEGs) after hesperetin treatment. E A bubble plot illustrating the KEGG pathway enrichment of the DEGs after hesperetin treatment. The grouping of the GO annotation and KEGG pathways were carried out by STRING v11.5 (https://string-db.org/). Pathway FDR < 0.05. F A heatmap illustrating that hesperetin down-regulates the expression of a panel of SASP-related factors (Hes vs Veh, Fold change > 1.1 and p < 0.05). G Canonical pathway analysis by Ingenuity Pathway Analysis (IPA) software based on Hes-mediated transcriptome changes (Hes vs. Veh; pathway p-value < 0.05). Data are presented as mean ± SD. *p < 0.05; **p < 0.005 by Student’s t test. DEGs differentially expressed genes, FC fold change, Redox reduction–oxidation, SASP senescence-associated secretory phenotype, UPR unfolded protein response, UV ultraviolet

Article Snippet: The HEK001 (CRL-2404, ATCC) human keratinocyte cell line, which was obtained from an older human subject, was cultured in keratinocyte-serum free medium (Gibco, Carlsbad, CA, USA, 17005-042) supplemented with human recombinant EGF (5 ng/mL), gentamicin (10 mg/mL), and 2 mM L-glutamine.

Techniques: Software, Expressing

Journal: Allergy

Article Title: miR-10a-5p is increased in atopic dermatitis and has capacity to inhibit keratinocyte proliferation

doi: 10.1111/all.13849

Figure Lengend Snippet: miR-10a-5p inhibits cell cycle progression and proliferation of primary KCs. (A-E) KC were transfected with miR-10a-5p mimic (miR-10a-5p), inhibitor (LNA-miR-10a) or corresponding controls for 48 h (A-C, E) or 72 h (D) and indicated assays were performed. (C, E) Indicated cytokines were added 24 h after transfection for additional 24 h. (A) Cell cycle distribution was measured based on DAPI signal. (B) Cells were labeled with EdU followed by measurment of DAPI and EdU signals by flow cytometry. (D) ATP-based proliferation assay, non-transfected (NT) cells were included as an additional control expected to proliferate faster compared to transfected cells. (C, E, F) RT-qPCR analysis of transfected KCs (C, E) or skin biopsies (F) from lesional (AD L) and non-lesional (AD NL) skin from AD patients and control individuals. Data represent the mean ± SEM. Student’s t-test, *P < 0.05, **P < 0.01.

Article Snippet: Pooled normal primary human KCs (PromoCell, Heidelberg, Germany) were cultured as previously described 26 and transfected either with 30 nM of hsa-miR-10a-5p Precursor, mirVana™ miR-10a-5p Mimic or corresponding Negative Controls #1.

Techniques: Transfection, Labeling, Flow Cytometry, ATP Proliferation Assay, Quantitative RT-PCR

Journal: Allergy

Article Title: miR-10a-5p is increased in atopic dermatitis and has capacity to inhibit keratinocyte proliferation

doi: 10.1111/all.13849

Figure Lengend Snippet: miR-10a-5p inhibits the expression of genes associated with cell cycle regulation, cell adhesion and cytokine signalling in KCs. KCs were transfected either with control or miR-10a-5p mimic (miR-10a-5p) for 24 hours and then stimulated with IL-1β or left nonstimulated (NS) for the next 24 hours. (A-C) Relative miRNA (A) and mRNA (B, C) levels are shown compared to nonstimulated control-transfected cells. Data represent mean ± SEM, Student’s t-test, *P < 0.05, **P < 0.01. (D) Heatmaps of miR-10a-5p influenced genes from the selected functional groups. Log2 values of mRNA expression signals are mean-centered for each gene separately. Color scale from blue (lower) to yellow (higher) represents deviation from the mean (black). Predicted direct targets are designated with bold.

Article Snippet: Pooled normal primary human KCs (PromoCell, Heidelberg, Germany) were cultured as previously described 26 and transfected either with 30 nM of hsa-miR-10a-5p Precursor, mirVana™ miR-10a-5p Mimic or corresponding Negative Controls #1.

Techniques: Expressing, Transfection, Functional Assay

Journal: Allergy

Article Title: miR-10a-5p is increased in atopic dermatitis and has capacity to inhibit keratinocyte proliferation

doi: 10.1111/all.13849

Figure Lengend Snippet: HAS3 is novel direct target of miR-10a-5p. (A) KCs in reconstituted epidermis were stimulated with indicated cytokines for 24 h or left nonstimulated (NS) and subjected to RT-qPCR analysis. Data is shown compared to the mean expression of HAS3 in NS cells (=1). (B) RT-qPCR analysis of skin biopsies from lesional (AD L) and non-lesional (AD NL) skin from AD patients and control individuals. (C) Immunofluorescence and DAPI staining of L and NL skin sections from AD patients and controls. Bars correspond to 50 μm. (D) KC were transfected with miR-10a-5p mimic (miR-10a-5p) or control for 24 h before indicated cytokines were added for additional 24 h. (E) miR-10a-5p and the mutated binding sites (underlined). Positions indicate the distance from the beginning of HAS3 3’UTR. (F) The relative firefly luciferase (LUC) activity is normalized to the values of control-transfected cells (=1) (n=8). (A, B, D, F) Data represent the mean ± SEM. Student’s t-test, * P < 0.05, ** P < 0.01.

Article Snippet: Pooled normal primary human KCs (PromoCell, Heidelberg, Germany) were cultured as previously described 26 and transfected either with 30 nM of hsa-miR-10a-5p Precursor, mirVana™ miR-10a-5p Mimic or corresponding Negative Controls #1.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Transfection, Binding Assay, Luciferase, Activity Assay