Role of Notch ligands in the effect of BMS and SS on DC differentiation. (A) Expression of Notch ligands in cultured BMS. Whole-cell lysates of BMS (2-week culture) were prepared and used to determine the expression of Jagged-1 and Delta-1 by Western blot. (B) RNA samples were extracted from BMS; cDNA was synthesized and used to determine expression of Jagged-1 and Delta-1. The number of gene copies per cell was determined using Jagged-1 and Delta-1 plasmids as described in “Materials and methods.” The Jagged-1–Delta-1 ratio was presented based on copy number per cell. (C) Expression of Jagged-1 and Delta-1 in 3T3 cell lines expressing Jagged-1 or Delta-1, BMS, and SS. Whole-cell lysates of 2-week cultured BMS or SS were prepared and used to determine the expression of Jagged-1 and Delta-1 by Western blotting as described in “Materials and methods” using indicated antibodies. BMS indicates bone marrow stroma; and SS, spleen stroma. Quantification was performed using densitometry. Levels of proteins were quantitated by using UN-Scan-IT software (Silk Scientific, Orem, UT). Expression in each sample was normalized for β-actin and was expressed as an arbitrary unit. It was calculated as follows: (intensity of Jagged-1 or Delta-1 bands)/(intensity of β-actin bands) × 100. (D) Expression of Jagged-1 and Delta-1 in bone marrow, spleens, and lymph nodes. RNA was extracted from tissues, and gene expression was evaluated as described in Figure 4B. (E) 3T3-Jagged-1 and 3T3-Delta-1 cells were mixed together at indicated ratios and then used to form a monolayer in 24-well plates. HPCs were isolated from bone marrow and cultured on a monolayer of fibroblasts in the presence of GM-CSF as described in the legend to Figure 1. Cells were collected on day 6 and analyzed by flow cytometry. Only CD45⁺ hematopoietic cells were analyzed, and the proportion of CD11c⁺ DCs was calculated. M indicates cells cultured on control 3T3-MSCV fibroblasts; D, cells cultured on Delta-1 fibroblasts; D/M, mixture of 3T3-Delta-1 and control 3T3-MSCV fibroblasts; and D/J, mixture of 3T3-Delta-1 and 3T3-Jagged-1 cells. (F) BMS cells were transfected with Jagged-1 or control siRNA. Twenty-four hours later, whole-cell lysates were prepared and the level of Jagged-1 in the cells was evaluated in Western blotting. The membranes were also probed with anti–Delta-1 antibody to assess potential off-target effect of siRNA. (G) HPCs were cultured on a monolayer of BMS transfected with Jagged-1 or control siRNA as well as on the monolayer of SS for 7 days in the presence of 20 ng/mL GM-CSF. Cells were transferred every 2 days on freshly prepared BMS. On day 7, cells were collected, labeled with different antibodies, and analyzed by flow cytometry. Only hematopoietic CD45.2⁺ cells were evaluated. Average ± SE of 3 experiments is shown. (H) CD45.2⁺ cells from the experiments described in Figure 4F were isolated using magnetic beads and cultured with allogeneic lymph node cells at different ratios. Cell proliferation was measured in triplicate using [³H]-thymidine uptake. Data represent the average ± SE of 3 experiments.