Browsing by Subject "Immunofluorescence"

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Open Access
Nuclear exclusion of p33ING1b tumor suppressor protein: explored in HCC cells using a new highly specific antibody
(Mary Ann Liebert, Inc, 2009) Sayan, B.; Emre, N. C. T.; Irmak, M. B.; Ozturk, M.; Cetin Atalay, R.
Mouse monoclonal antibodies (MAb) were generated against p33ING1b tumor suppressor protein. 15B9 MAb was highly specific in recognizing a single protein band of ∼33 kDa endogenous p33ING1b protein from HCC cell lines and normal liver tissue by Western blot analysis and by immunoprecipitation. Although p33ING1b mutations are rarely observed in cancer, differential subcellular distribution and nuclear exclusion of p33ING1b were reported in different cancer types. Therefore we analyzed the expression and subcellular localization of p33ING1b in HCC cell lines using 15B9 MAb. So far, p33ING1b mutations or differential subcellular localization are not reported in HCC. In this study, by indirect immunofluorescence using MAb 15B9, we demonstrate that nuclear localization of p33ING1b was highly correlated with well-differentiated HCC cell lines whereas poorly differentiated HCC cells have nuclear exclusion of the protein. Moreover no association was observed between differential subcellular localization of p33ING1b and p53 mutation status of HCC cell lines. Hence our newly produced MAb 15B9 can be used for studying cellular activities of p33ING1b under normal and cancerous conditions. © Copyright 2009, Mary Ann Liebert, Inc.
Open Access
The primary cilia of the gastrointestinal tract in homeostasis and disease at the single-cell level
(2021-07) Esen, Deniz
The gastrointestinal tract is regularly renewed by stem cells which divide and differentiate into functionally and morphologically distinct cell types. Several key pathways, such as Wnt, Hedgehog and Bmp, regulate the cell fate. However, it remains a mystery how the associated signaling molecules are relayed between cells to coordinate stemness and differentiation cues. Primary cilia are small antenna-like organelles that harbors many receptors for these pathways. Here public single-cell RNA sequencing data is re-analyzed to show that primary cilia expression is heterogeneous in the intestinal mesenchyme and liver. Presence of primary cilia is also validated using immunofluorescence in the stroma and muscle cells of the mouse colon, using known markers of the primary cilia. Acot7 is identified as a primary cilium associated marker and found to be expressed in myenteric ganglia. In mice challenged with DSS to model ulcerative colitis, primary cilia are observed more abundant as the area covered by crypt structures become reduced due to the loss of epithelium. Acot7 expressing ganglial cells were observed more frequently and displayed morphological differences. Additionally, mice fed a high-fat diet over 16 weeks had shortening of the colon crypts and an increase in the primary cilia. This work suggests that primary cilia exist in the gastrointestinal tract during homeostasis and participate in inflammation and diet-based adaptations.