To further investigate the HSP signaling

To further investigate the HSP90 signaling pathway we inhibited the downstream molecule PI3K, which is involved in cell migration and ARQ 621 signaling (Fukata et al., 2003; Yoneda et al., 2005; Zhu et al., 2009b). Migration signaling involves the Rac GTPase interacting protein, which has been implicated in cilia elongation (Kim et al., 2010). Our data supported this as PI3K inhibition, alone or in combination with SFM treatment, resulted in increases in ciliation (Fig. 7A) and significantly affected cilia length. Interestingly, combining PI3K inhibition with SFM treatment produced effects that were oxygen tension dependent (Fig. 7B). Overall the effect of inhibitors used in this study produced small but significant changes in cilia length, comparable to other reports (Ghossoub et al., 2013; Hori et al., 2014; Prodromou et al., 2012; Wann et al., 2013; Yang et al., 2013).
Author contributions The following are the supplementary data related to this article.
Acknowledgments JALB and TS are funded by Systems Biology Ireland through Science foundation Ireland grant 06/CE/B1129. We would like to thank Professor Ciaran Morrison (Centre for Chromosome Biology) for the gift of Smo and Gli2 antibodies, Dr Aline Morrison (REMEDI) for providing the BMSCs and Dr Shirley Hanley (REMEDI) for the assistance with FACS. We would also like to thank Dr Róisín Dwyer and Dr Emer Bourke for critical discussion of the manuscript.
Introduction
Material and methods
Results
Discussion We have generated and characterized a Nanog-inducible mouse with high Nanog expression levels in most major organs. Contrary to what we expected, prolonged Nanog expression led to phenotypic changes that were restricted to the small intestine and colon, leaving other major organs unaffected. Although Nanog does not play a role in the formation of the gastrointestinal tract during embryonic development, its overexpression during adulthood leads to hyperplasia of the epithelium and abnormal morphology of the mucosal architecture (Fig. 2A). Hyperplastic outgrowths were always observed but they never led to the formation of adenomas or carcinomas. To gauge whether mice that had not reached adulthood would show a stronger response to Nanog expression, newly weaned 3-week-old mice were analyzed. Though these mice were not able to withstand the treatment for longer than 2–3weeks, they showed signs of hyperplasia after only 7days. The villi of the small intestine doubled in length on day 7 and continued to grow until day 9, after which time the extension leveled off. Three weeks after withdrawal of dexamethasone and doxycycline, downregulation of Nanog expression and reduced villus length were once again observed, showing that the hyperplasia is dependent upon continuous Nanog expression (Fig. 2B). The colon also showed signs of hyperplasia after 9days of Nanog induction. As shown in Fig. 2C, the colonic mucosa, which has a typical flat morphology, developed outgrowths that were pathologically identified as hyperplasia of the colonic epithelium. Moreover, a reduction in the number of goblet cells within these outgrowths was observed by PAS staining. Forced expression of Nanog in NIH 3T3 cells and human ES cells has been shown to promote entry into the S-phase of the cell cycle, enhancing the rate of proliferation (Zhang et al., 2005, 2009). To determine whether Nanog influences the proliferation of the intestinal tract, a time course of BrdU incorporation was performed after 7days of Nanog overexpression. Indeed, 2h after BrdU administration, an additional 85% of cells in the crypts showed proliferation in response to Nanog treatment (Fig. 3C). This is an interesting finding, as the intestinal stem cells residing in the crypts are responsible for the repopulation of the villi with differentiated cells. The next time point, 48h post–BrdU administration, further showed that cells labeled with BrdU migrated faster up the crypt compartment in the Nanog-induced mice. The possibility that the observed signal was due to dormant epithelial cells that had initiated proliferation was excluded, as the BrdU-labeled cells were not scattered around the villi but were all in line with the natural migration pathway from the crypt up the villi. Furthermore, no proliferation of the typically dormant cells was observed between 2 and 48h (see representative example in Fig. 3B). These results clearly demonstrated that altered proliferation within the crypt compartment plays a dominant role in the development of the observed hyperplasia. The morbidity and deaths of 6-week-old mice upon Nanog induction might be caused by a combination of doxycycline/dexamethasone/ethanol treatment and malabsorption related to the intestinal phenotype.