Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: KLK5 up-regulates TSLP and proinflammatory molecules in keratinocytes. (A) NHKs were stimulated with 400 nM of recombinant KLK5 for 3 h. TSLP , ICAM1 , IL8 , and TNF -α transcripts are increased by seven-, five-, four-, and fivefold, respectively. No difference was observed for IL-1β , TARC , and MDC ( n = 5). (B) NHK transfected with 60 pmol PAR2 siRNA and stimulated with KLK5 showed PAR2 mRNA down-regulation (by 56%) 4 d after transfection. In PAR2 siRNA-transfected cells, KLK5 is less efficient in inducing TSLP , ICAM1 , IL8 , and TNF-α mRNA (48, 55, 60, and 62% of control, respectively; n = 4). (C) In cells treated with 10 µM NF-κB pathway inhibitor (BAY11-7082), KLK5 efficiency to up-regulate TSLP , ICAM1 , IL8 , and TNF-α mRNA is decreased by 81, 70, 59, and 40%, respectively ( n = 3). For each experiment, the mRNAs of interest were measured by quantitative RT-PCR. Each point is the mean of triplicate amplification for at least three independent experiments ( n ). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Recombinant, Transfection, Control, Quantitative RT-PCR, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: TSLP and proinflammatory molecules are induced by KLK5 in keratinocytes of NS patients. (A) Quantitative RT-PCR on cultured primary keratinocytes reveals that TSLP is significantly overexpressed by fivefold in three NS patients (NSK) compared with three healthy controls (NHK). ICAM1 , IL8 , TNF-α , TARC , and MDC mRNA are overexpressed in two out of three NSKs but not IL-1β mRNA. Each point represents the mean of two independent experiments for each individual. *, P < 0.05. (B) NHK and NSK were treated with 10 µg/ml brefeldin A, and TSLP was detected by immunofluorescence (green). Blue staining is DAPI. Pictures are representative of two independent brefeldin A treatments, each realized on two NHKs and two NSKs. (C and D) NSKs were transfected with KLK5 siRNA and its efficient down-regulation was confirmed by quantitative RT-PCR, Western blotting (laminin 5 used as loading control), and ELISA, 48 and 72 h after transfection. Results are representative of three independent experiments. (E) KLK5 knockdown induces a decrease of TSLP measured by quantitative RT-PCR. ICAM1 , IL8 , and TNF-α mRNAs were less efficiently reduced. Data are the mean ± SD of one experiment, which is representative of three independent experiments. Bars, 49 µm

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Quantitative RT-PCR, Cell Culture, Immunofluorescence, Staining, Transfection, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: KLK5 triggers proinflammatory and proallergic microenvironment independently of external stimuli. Lack of KLK5 inhibition by LEKTI initiates proinflammatory and proallergic cascades independently of environmental factors. In LEKTI-deficient keratinocytes, hyperactive KLK5 directly induces TSLP, IL8, and TNF-α overexpression through PAR2 and NF-κB pathway activation. Unregulated KLK5 also degrades desmosomes at the interface between the GR and the SC leading to defective SC adhesion and IL-1β, IL8, and TNF-α secretion by mechanically stressed keratinocytes. In addition, all these cytokines could induce TARC and MDC chemokines secretion by keratinocytes and dermal fibroblasts. These proinflammatory mediators trigger eosinophilic and mast cell recruitment and activation. TSLP has been reported to activate resident LCs, which migrate to draining lymph nodes and promote the differentiation of naive T cells (Th0) into Th2 cells. Collectively, activated keratinocytes together with eosinophilic and mast cells induce pro-Th2 microenvironment favoring the development of an AD-like phenotype.

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Inhibition, Over Expression, Activation Assay

Journal: Nature communications

Article Title: Genome-wide compendium and functional assessment of in vivo heart enhancers.

doi: 10.1038/ncomms12923

Figure Lengend Snippet: Figure 4 | Cardiac enhancers are required for normal expression of heart disease-associated genes. Knockout analysis of enhancers mm77 (a,c,e,g,h) and mm771 (b,d,f,i,j). (a,b) Representative transgenic reporter assay results. Heart activity is indicated by red arrows. Numbers indicate reproducibility over total transgenic embryos. Gene models not drawn to scale. LOF: loss-of-function, GOF: gain-of-function. (c,d) Chromosome-wide mRNA expression changes in E11.5 whole heart. Points indicate individual genes, with red indicating statistically significantly differences after FDR correction (Po0.01 using an FDRo5%, see the ‘Methods’ section for details). Dashed grey line indicates position of enhancer. Mb: megabases. (e,f) Normalized mRNA levels measured by quantitative RT-PCR in E11.5 hearts. Boxplots indicate median and quartile values for each data set; points indicate outliers. (g–j) Representative western blot images (g,i) and normalized protein levels (h,j) in wild-type ( þ ) and homozygous null ( ) samples. Gapdh was used as a loading control, MW, molecular weight. (g,i). Bars indicate group means, and points represent biological replicates (h,j). For (e,f,h,j): values were normalized to the wild-type mean, and P values were calculated using a one-tailed t-test.

Article Snippet: RNA sequencing libraries were made using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat kit (Illumina) or the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to manufacturer instructions.

Techniques: Expressing, Knock-Out, Transgenic Assay, Reporter Assay, Activity Assay, Quantitative RT-PCR, Western Blot, Control, Molecular Weight, One-tailed Test

Journal: Genes

Article Title: Chromosome 4 Duplication Associated with Strabismus Leads to Gene Expression Changes in iPSC-Derived Cortical Neurons

doi: 10.3390/genes16010080

Figure Lengend Snippet: Heatmap of the DEGs in neuroprogenitors ( A ) and cortical neurons ( B ) and validation by qPCR ( C ). ( A ) Heatmap showing the expressions (z-scores) of differentially expressed genes in neuroprogenitor cells between controls and cells with duplication (fourth independent differentiations). ( B ) Heatmap showing the expressions (z-scores) of differentially expressed genes in cortical neurons between controls and cells with duplication, a pink brain next to the gene name represents a neuronal/synapse related function and a blue brain next to the gene name represents genes associated with neurodevelopmental disorders. ( C ) qRT-PCR validation from randomly selected upregulated and downregulated genes; total RNA from genotypes at day 14 and day 63 was used to analyze expression levels, and data were normalized using TBP as the reference gene.

Article Snippet: Then, 200 ng of high-quality RNA from each sample was used for total RNA sequencing library preparation with the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Plus for Human/Mouse/Rat (Cat. No. RS-122-2201 (Illumina Inc., San Diego, CA, USA), following the manufacturer’s instructions.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing

Journal: Diabetes

Article Title: MicroRNA-29 fine-tunes the expression of key FOXA2-activated lipid metabolism genes and is dysregulated in animal models of insulin resistance and diabetes.

doi: 10.2337/db13-1015

Figure Lengend Snippet: Figure 1—Hepatic miR-29 levels are upregulated in diet-induced insulin resistance in mice. A: Fasting blood glucose levels of C57BL/6J female mice on HFD for 16 weeks (n = 3) and matched LFD for 16 weeks (n = 3) are shown. B: miRNA expression levels (RPMM, reads per million mapped reads) based on deep sequenc- ing analysis of small RNAs from the livers of HFD-fed (n = 2) and LFD-fed (n = 2) C57BL/6J female mice are shown. Each circle rep- resents a miRNA that is expressed at RPMM >100 in at least one murine liver sample. Dashed blue lines represent 1.5-fold difference in expression between HFD-fed and LFD-fed mice. C: Relative lev- els (based on sequencing) of miR-29a, miR-29b, and miR-29c in the livers of HFD-fed (n = 2) and LFD-fed (n = 2) C57BL/6J female mice are shown. D: Relative levels (based on RT-qPCR) of miR-29a, miR- 29b, and miR-29c in the livers of HFD-fed (n = 3) and LFD-fed (n = 3) C57BL/6J female mice are shown. P values were calculated according to the one-tailed unpaired Student t test. a.u., arbitrary unit. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Small RNA libraries (n = 2 for each of HFD-fed and LFD-fed mice) were generated using the Illumina TruSeq Small RNA library preparation kit.

Techniques: Expressing, Sequencing, Quantitative RT-PCR, One-tailed Test