Notch activation in OB, SC, and LSK cocultures. (A) Endogenous expression of components of the Notch pathway. Quantitative RT-PCR was performed on cDNA generated from mRNA derived from sorted LSK cells, cultured SCs, and freshly isolated 2-day C OBs (performed in triplicate for each sample). Bar graphs represent ratio of each specific transcript to glyceraldehyde-3-phosphate dehydrogenase. Expression of each transcript in OBs and SCs was expressed as fold change relative to the expression of that transcript in LSK cells, which was normalized to 1. (B) Quantitative RT-PCR data from cells isolated from cocultures. Cocultures were established with SCs from C57Bl/6 GFP mice (CD45.2), OBs (2-day C) from C57Bl/6 mice (CD45.2), and LSK cells from BoyJ mice (CD45.1). On day 7, cells were harvested and stained with PE-CD45.1. mRNA was prepared from GFP-CD45.1⁺ cells (LSK progeny only) and analyzed. Bar graphs show fold increase in the expression of the indicated genes in LSK cultured alone (normalized to 1 in cultures without GSI) or with other cell types shown for each condition in parentheses. Quantitative RT-PCR was performed in triplicates for each sample and each condition. Data are representative of 2 independent experiments with similar results. GSI was added at 10nM on day 0 and replenished twice during the next 7-day culture period. The legend shown in the plot of Deltex in panel B applies to all other plots in the figure (N1, N2, J1, J2, and Hes1).