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

Journal: Frontiers in Immunology

Article Title: ULK1 Inhibition as a Targeted Therapeutic Strategy for Psoriasis by Regulating Keratinocytes and Their Crosstalk With Neutrophils

doi: 10.3389/fimmu.2021.714274

Figure Lengend Snippet: Inhibition of ULK1 suppresses proliferation and promotes apoptosis of keratinocytes in vitro . (A) Immunoblot of HaCat keratinocytes 24 hours after cocultured with DMSO, 5 µM or 10 µM SBI-0206965 (SBI). (B) Cell cycle analysis of HaCat keratinocytes treated with DMSO or 10 µM SBI for 24 hours. The bar graph shows the percentage of cell population in each phase of cell cycle. (C) Apoptosis of HaCat keratinocytes 24 hours after serum deprivation in the presence of DMSO or 10 µM SBI. (D) mRNA expression of psoriasis-related inflammatory mediators in HaCat keratinocytes cocultured with DMSO or 10 µM SBI for 24 hours. (E) mRNA and protein expression of ULK1 in HaCat keratinocytes transfected with negative control-siRNA(NC-siRNA) and ULK1-siRNA. (F) Cell cycle analysis of HaCat keratinocytes 72 hours after transfection with NC-siRNA or ULK1-siRNA. (G) Apoptosis of transfected HaCat keratinocytes 24 hours after serum deprivation. (H) mRNA expression of psoriasis-related inflammatory mediators in HaCat keratinocytes transfected with NC-siRNA or ULK1-siRNA for 72 hours. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Primary keratinocytes (PCS-200-010, ATCC) were cultured in Dermal Cell Basall Medium (PCS-200-030, ATCC) supplemented with Keratinocyte Growth Kit (PCS-200-040, ATCC), 10U/mL of penicillin and 10μg/mL of streptomycin and 25ng/mL of amphotericin (03-033-1B, BI).

Techniques: Inhibition, In Vitro, Western Blot, Cell Cycle Assay, Expressing, Transfection, Negative Control

Journal: Frontiers in Immunology

Article Title: ULK1 Inhibition as a Targeted Therapeutic Strategy for Psoriasis by Regulating Keratinocytes and Their Crosstalk With Neutrophils

doi: 10.3389/fimmu.2021.714274

Figure Lengend Snippet: Inactivation of ULK1 by SBI suppressed the inflammation in keratinocytes stimulated by neutrophil. (A) mRNA expression of psoriasis-related inflammatory mediators by keratinocytes stimulated with neutrophils isolated from healthy donors (HC) or (B) psoriasis patients in the presence of 10 µM DMSO or SBI for 10 hours. Data are representative of three independent experiments. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Primary keratinocytes (PCS-200-010, ATCC) were cultured in Dermal Cell Basall Medium (PCS-200-030, ATCC) supplemented with Keratinocyte Growth Kit (PCS-200-040, ATCC), 10U/mL of penicillin and 10μg/mL of streptomycin and 25ng/mL of amphotericin (03-033-1B, BI).

Techniques: Expressing, Isolation

Journal: Frontiers in Immunology

Article Title: ULK1 Inhibition as a Targeted Therapeutic Strategy for Psoriasis by Regulating Keratinocytes and Their Crosstalk With Neutrophils

doi: 10.3389/fimmu.2021.714274

Figure Lengend Snippet: Autophagy inhibitors fail to fully replicate the effect of ULK1 inhibition on keratinocyte. (A–C) Expression of p62 and LC3 I/II in HaCat keratinocytes 24 hours after incubation with SBI0206965 (SBI) (A) or chloroquine (B) or 3-methyladenine(3-MA) at indicated concentration (C) . (D) Cell cycle analysis and (E) apoptosis of keratinocytes after 24 hours with the treatment of 10 µM chloroquine or 5 mM 3-MA. (F) mRNA expression of psoriasis-related inflammatory mediators by keratinocytes cocultured with neutrophils from healthy donors in the presence of 5 mM 3-MA. Data are representative of three independent experiments. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Primary keratinocytes (PCS-200-010, ATCC) were cultured in Dermal Cell Basall Medium (PCS-200-030, ATCC) supplemented with Keratinocyte Growth Kit (PCS-200-040, ATCC), 10U/mL of penicillin and 10μg/mL of streptomycin and 25ng/mL of amphotericin (03-033-1B, BI).

Techniques: Inhibition, Expressing, Incubation, Concentration Assay, Cell Cycle 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: 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: 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