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Nod-Like Receptor Pyrin domain-containing protein 6 (NLRP6), a member of the Nucleotide-oligomerization domain-Like Receptor (NLR) family of proteins, assembles together with the ASC protein to form an inflammasome upon stimulation by bacterial lipoteichoic acid and double-stranded DNA. Besides its expression in myeloid cells, NLRP6 is also expressed in intestinal epithelial cells where it may contribute to the maintenance of gut homeostasis and negatively controls colorectal tumorigenesis. Here, we report that NLRP6 is very faintly expressed in several colon cancer cell lines, detected only in cytoplasmic small dots were it colocalizes with ASC. Consequently, it is very hardly detected by standard western-blotting techniques by several presently available commercial antibodies which, in contrast, highly cross-react with a protein of 90kDa that we demonstrate to be unrelated to NLRP6. We report here these results to caution the community not to confuse the 90kDa protein with the endogenous human NLRP6. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) skin is characterized by over-expression of interleukin (IL)-4, IL-13 and IL-25. When methyl-β-cyclodextrin (MβCD) treatment preceded exposure to these interleukins, combination of both treatments was found to mimic hallmarks of AD in vitro, such as barrier weakening, histological alterations and typical signaling responses in a reconstructed human epidermis (RHE). However, the respective role of each IL and whether any of them is critical when combined with MβCD treatment was unknown. Therefore, this work aimed to distinguish RHE responses after exposure to MβCD and each one of the three IL reported to mimic typical features of AD. IL-4 incubation preceded by MβCD was found responsible for altered histology, as well as for barrier alterations, evidenced by electrical resistance and dye permeation measurements. This combination further decreased loricrin (LOR) immunoreactivity, whereas mainly IL-25, combined to MβCD treatment, was able to downregulate filaggrin (FLG) mRNA level. Carbonic anhydrase II (CA2) and hyaluronan synthase 3 (HAS3), two other markers up-regulated in AD, were also induced when MβCD treatment was followed by IL-4, whilst the expression of neural epidermal growth factor-like 2 (NELL2) was up-regulated by paired IL-4 and IL-13. In conclusion, multiple features of AD were found in this in vitro model mainly when treatment of RHE by IL-4 was conducted after preliminary MβCD incubation. [ABSTRACT FROM AUTHOR]

The article discusses research on the use of sufficient reference genes for effective interpretation of data about gene expression in skin diseases. Topics cited include the different tools for mRNA analysis such as Northern blot hybridization and reverse-transcription quantitative polymerase chain reaction, precautionary measures in conducting gene expression analysis in heterogeneous cell populations in cultures and tissues, the keratinization process, and keratinocyte proliferation.