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: Frontiers in Pharmacology

Article Title: Syzygium aqueum : A Polyphenol- Rich Leaf Extract Exhibits Antioxidant, Hepatoprotective, Pain-Killing and Anti-inflammatory Activities in Animal Models

doi: 10.3389/fphar.2018.00566

Figure Lengend Snippet: Effect of Syzygium aqueum extracts on human keratinocytes. (A) Biocompatibility of S. aqueum extract in dose (25–200 μg/mL) and time (24–48 h) manner. Survival rate of the cells was assessed by the MTT assay. (B) ROS production in cells treated or untreated with theextract (100 μg/mL) and then irradiated by 100 J/cm 2 UVA (+) or not (–). (C) Total GSH levels determined by DTNB assay. Results expressed as mean ± SD of three assays. Significant ∗ p < 0.01; ∗∗ p < 0.001 compared to untreated control. Analysis performed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. (D) Immunoblotting bands of the oxidative stress activated protein, p38. An equal amount (100 μg) of total lysate from each sample was resolved by SDS-PAGE with GAPDH as a control.

Article Snippet: Human epidermal keratinocytes (HaCaT), provided by Innoprot (Biscay, Spain), were cultured as described in .

Techniques: MTT Assay, Irradiation, DTNB Assay, Western Blot, SDS Page

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