Gene expression data were normalized with www.selleckchem.com/products/BI6727-Volasertib.html Rps6 (Mm02342456_g1) (Applied Biosystems). RESULTS Discrete Levels of Sox9-EGFP Transgene Expression Mark Four Different Cell Populations that Exhibit Distinct Gene Expression Signatures by Microarray We first aimed to confirm and strengthen previously reported evidence that IEC populations expressing different levels of the Sox9-EGFP transgene are enriched for phenotypically distinct cells (17, 21). These prior studies demonstrated that the Sox9-EGFP transgene is expressed at low/moderate levels (Sox9-EGFP Low; Fig. 3A) in cells with ISC characteristics (21), intercalated between Sox9-EGFP Negative Paneth cells (17), and at higher/more intense levels (Sox9-EGFP High; Fig. 3A) in cells that colabel with the EEC marker ChgA (17, 21). As illustrated in Fig.

3A, this expression pattern is maintained in independent animals derived from breeding this model across multiple generations. In addition, Fig. 3A demonstrates that a short 90-min pulse of the S-phase marker EdU predominantly labels cells located higher in the crypt than Sox9-EGFP Low and High cells where rapidly dividing progenitors reside, and these cells express very low or what have been termed Sublow levels of Sox9-EGFP (Sox9-EGFP Sublow) (21). Fig. 3. Sox9-EGFP transgene marks 4 different cell populations that exhibit distinct gene expression signatures. A: confocal microscopy on a jejunal crypt from a nonirradiated Sox9-EGFP mouse illustrates that the Sox9-EGFP transgene (green) is expressed at different …

Illustrative flow cytometry data demonstrate the gradient of expression of the Sox9-EGFP transgene in dissociated single cell preparations of IECs obtained from nonirradiated Sox9-EGFP mice (Fig. 3B). Based on distinct levels of Sox9-EGFP, four different cell populations, i.e., Sox9-EGFP Negative, Sublow, Low, and High cells, were distinguished and isolated by FACS by using gating procedures previously reported (21) (Fig. 3B; also see Fig. 1A). Gene microarray was performed on Sox9-EGFP Negative, Sublow, Low, and High cells isolated from jejunum of four independent animals. Unsupervised principal component analysis and hierarchical clustering of gene expression data clearly demonstrated that Sox9-EGFP Negative, Sublow, Low, and High cells each exhibit a unique gene expression signature, which is consistent across individual mice (Fig. 3, C and D). Real-time quantitative PCR analysis validated that the expression profile of EGFP in FACS isolated cells matched with the four different Sox9-EGFP groups (Fig. 3E). Also, endogenous Sox9 expression paralleled EGFP mRNA abundance (Fig. 3F), confirming that the Sox9-EGFP reporter gene faithfully recapitulates Entinostat expression patterns of endogenous Sox9 (17, 21, 49).