Supplementary MaterialsSupplementary material JHC702586_Supplemental_Figures. or repelling (OR 1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs.? values of less than 0.05 were considered significant. Results pY1798 Revealed Epithelial Cells Closely Resembling TCs in the Mammalian Gut We first attempted IHC of mouse jejunum using newly developed site-specific and phosphorylation-statusCspecific antibodies against human Girdin tyrosine-1798 (pY1798 antibodies).20 We previously verified the specificity of the pY1798 antibodies using (1) dot-blot assay with phospho/unphosphorylated peptides, (2) western blot of HEK293FT cells transfected with a Girdin expression vector with/without the tyrosine-phenylalanine substitution at 1798 (Y1798F), (3) immunofluorescence of kinase-stimulated cell-lines, and (4) IHC of Girdin wild-type/knockout mouse brains.20 These pY1798 antibodies identified sporadic strongly stained epithelial cells with unusual morphological characteristics, including spool-shaped somas with a single mass of signal condensation at each lumenal tip (Fig. 1A, upper). pY1798-positive epithelial cells were (1) found throughout the entire small intestine, Clofarabine ic50 (2) widely scattered from crypt to villus tip, and (3) never adjacent to each other. pY1798-positive cells accounted for about 1% Clofarabine ic50 of the entire epithelium in the mouse jejunum, in which the percentage showed regional variation as well as individual animal variability. Regarding the subcellular localization, the pY1798 staining was not restricted to the apical microvilli, but was also present in all over the cytoplasm, including the apical cytoplasm (above and around the nucleus) and the sub-membranous area. pY1798 staining was barely seen within the nuclei. Besides the epithelium, pY1798-positive cells were sporadically observed in the lamina propria. The appearance of pY1798-positive epithelial cells in the mouse jejunum was indistinguishable from previously reported TCs labeled with Cox2 (Fig. 1A, lower). In contrast to the clear staining obtained with pY1798 (post-absorbed with unphosphorylated Y1798 peptide), pre-absorbed pY1798 antibodies did not specify TC-like epithelial cells, and instead labeled a broad spectrum of cells, indicating the widespread presence of unphosphorylated Girdin at Y1798 in enterocytes of the mouse jejunum (Fig. 1B). Open in a separate window Figure 1. pY1798 reveals epithelial cells closely resembling tuft cells (TCs) in the mammalian gut. (A) Immunohistochemistry of mouse jejunum with anti-Girdin phospho-Y1798 (pY1798) antibodies or with cyclooxygenase-2 (Cox2) antibodies. The boxed areas in the low-magnification images (scale bar, 100 m) were magnified, rotated, and shown on each right side. Red dots represent positive epithelial cells. Immunohistochemistry of mouse jejunum with pY1798 antibodies pre-absorption (B, upper), or post-absorption (B, lower) using unphosphorylated Y1798 peptides (scale bars, 10 m). Immunohistochemistry of mouse (C) and human (D) gastrointestinal tracts with LATS1 pY1798 antibodies (scale bars, 10 m). IHC of multiple organs (stomach, duodenum, jejunum, ileum, colon, and gallbladder) from mouse and human for pY1798 also revealed sporadic epithelial cells with similar morphological characteristics of TCs (Fig. 1C and ?andD).D). pY1798 signals were not observed in the esophagus, suggesting the specific distribution of pY1798-positive epithelial cells within columnar epithelia. In previous publications, intestinal TCs were often drawn in illustrations as epithelial cells with somas slightly deviated toward the lumen.5 TCs double-positive for pY1798 and villin had a similar deviation tendency in all tested tissues in human and Clofarabine ic50 mouse to a varying degree (Fig. 1C and ?andD,D, Supplemental Fig. 3). Even in the small intestine of global Girdin knockout mice, TCs were labeled with known TC-markers (lectin UEA-I, or Cox2; Supplemental Fig. 1A). In contrast, pY1798-positive epithelial cells were completely invisible in knockout mouse organs (duodenum, jejunum, ileum, and gallbladder; Supplemental Fig. 1B), clearly indicating that the pY1798 antibody specifically recognizes mouse Girdin gene products. Validation of pY1798 as a TC-Marker by Transmission Electron Microscopy Despite the lack of consensus molecular markers to define TCs, the earliest TC researchers described.