Altered gene expression and protein levels in stem cells and progenitors lacking microenvironmental Ptn. (A) Gene expression analysis in sorted LSK cells from Ptn^−/− BM, from cocultures with Ptn^KD (n = 5), or from cocultures on primary Ptn^−/−stromal cells (n = 3). Gene expression was normalized relative to the housekeeping gene Gorasp2. Shown is the ratio of the normalized gene expression to Ptn^+/+, pLKO.1, and primary Ptn^+/+ stroma. (B) Ccnd1 protein levels in 3-day cocultures on Ptn^KD or pLKO.1 UG26-1B6 obtained by intracellular flow cytometry. Shown is a representative example. (C) Percentages of Ccnd1^high cells in LSK cells recovered from 3-day cocultures on pLKO.1⁻ or Ptn^KD UG26-1B6 cells. (D) Cebpa protein levels in LSK cells sorted from the BM of Ptn^+/+ and Ptn^−/− mice. (E) Pixel quantitation of stained LSK cells from Ptn^−/− mice (white bars, n = 3), cocultures on Ptn^KD (gray bars, n = 6), or primary Ptn^−/− stromal cells (black bars, n = 3). Shown is the ratio of pixels to Ptn^+/+, pLKO, or WT stroma, respectively. (F-G) Similar measurements of Ccnd1 protein levels in LSK cells from the BM of Ptn^−/− mice, cocultures on primary Ptn^−/− stromal cells. Cocultures on Ptn^KD stroma could not be analyzed. All values are mean ± SEM; statistics were performed with the paired Student t test, *P < .05.