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

Journal: Oncology Letters

Article Title: TRPC7 facilitates cell growth and migration by regulating intracellular Ca 2+ mobilization in lung adenocarcinoma cells

doi: 10.3892/ol.2023.13678

Figure Lengend Snippet: Effect of TRPC7 on cell proliferation in lung adenocarcinoma. (A) Immunoblot detection of the protein expression level of TRPC7 in lung cell lines. Normal lung cell lines were BEAS-2B (human bronchial epithelium), BES-1A1.6 (human bronchial epithelium) and IMR-90 (human lung fibroblast). Squamous cell carcinoma cell lines were H125 and H520. Adenocarcinoma cell lines were A549 and H1299. KC were used as TRPC7-positive control cells. (B) Further comparison of endogenous TRPC7 protein expression levels among normal lung, squamous cell carcinoma and adenocarcinoma cell lines using LC/MS. Quantification of the TRPC7 expression analyzed using unpaired Student's t-test. Data were presented as mean ± SD. Effect of (C) TRPC7 knockdown and (D) TRPC7 overexpression on H1299 and BEAS-2B cell proliferation (analyzed using unpaired t-test), respectively. Reverse transcription-quantitative PCR and immunoblotting analysis demonstrated that TRPC7 mRNA and protein expression levels were (E) reduced in H1299 and (F) overexpressed in BEAS-2B cells (analyzed using unpaired t-test). The percentage of (G) TRPC7 knockdown H1299 and (H) TRPC7-overexpressing BEAS-2B cells in the G0/G1, S and G2/M phases (analyzed using unpaired Student's t-test; n=3). Data were presented as mean ± SD for all experiments. *P<0.05, **P<0.01 and ***P<0.001. KC, primary normal human epidermal keratinocytes; LC/MS, liquid chromatography tandem mass spectrometry; OE, overexpression; si, small interfering RNA; TRPC7, transient receptor potential canonical 7.

Article Snippet: H125 and the primary normal human epidermal keratinocytes (cat. no. C-12005) were purchased from Creative Biolabs, Inc. and PromoCell GmbH., respectively.

Techniques: Western Blot, Expressing, Positive Control, Comparison, Liquid Chromatography with Mass Spectroscopy, Knockdown, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Liquid Chromatography, Mass Spectrometry, Small Interfering RNA