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

Journal: Frontiers in Immunology

Article Title: Sulfotransferase SULT2B1 contributes to the epithelial–immune microenvironment homeostasis in imiquimod-induced psoriatic dermatitis

doi: 10.3389/fimmu.2025.1632426

Figure Lengend Snippet: T-helper (Th)-1 cytokines promote SULT2B1 mRNA expression in human keratinocytes. (A) Epidermal CS levels in healthy controls (n = 30) and patients with the indicated skin diseases [seborrheic keratoses (n = 9), actinic keratoses (n = 10), tinea corporis (n = 3), atopic dermatitis (non-lesional, n = 9; lesional, n = 10), and psoriasis (non-lesional: n = 16; lesional: n = 37)] quantified via supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS; one-way ANOVA, followed by Dunnett’s multiple-comparison test). (B, C) Real-time PCR analysis of SULT2B1 gene levels in undifferentiated (B) or differentiated (C) normal human epidermal keratinocytes (NHEKs) treated with two concentrations of the indicated cytokines (n = 6/group; one-way ANOVA, followed by Dunnett’s multiple-comparison test). Data were obtained from six (B, C) independent experiments, and graphs are represented as the mean ± SD. ** P < 0.01 and *** P < 0.001; ns, not significant.

Article Snippet: Primary normal human epidermal KCs (NHEKs) isolated from adult skin (#CA10205a; Cell Applications, San Diego, CA, USA) were cultured in T-75 flasks with the Human EpiVita Serum-Free Growth Medium Kit for Adult Cells (#A141K500a; Cell Applications) containing 0.06 mM Ca 2+ , according to the manufacturer’s instructions.

Techniques: Expressing, Supercritical Fluid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Comparison, Real-time Polymerase Chain Reaction