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