Figure 5. In vitro HSPC-biased activity by high-affinity monomer S4-3a compared to wild-type SCF.

(A) Ex vivo CFU-GM colony formation assay. Sorted mouse LSK HSPCs were cultured in semi-solid methylcellulose medium with 100 ng/ml recombinant mouse IL-3 and the indicated concentration of SCF variants. The number of CFU-GM colonies were scored after a 7-day period and normalized to the top data point in the 5000 ng/ml SCF group. (B) Ex vivo proliferation of purified LSK HSPCs from mouse bone marrow. Cells were cultured on irradiated OP9 stromal cells for 6 days in the presence of titrating concentrations of SCF variants, and the total number of hematopoietic cells generated was quantified. (C) β-hexosaminidase release by human PBCMCs treated with anti-IgE alone or with anti-IgE together with SCF or S4-3a at indicated concentrations (ng/ml) for 30 min in vitro. See also Figure S4. (D) Dose-response curves of IL-6 release by mouse BMCMCs stimulated for 6 h with SCF variants in vitro. (A and C) Data represent mean ± SEM and are pooled from at least two independent experiments with a total of n 6. *p<0.05, **p<0.01, ***p<0.001, and ns = not significant (i.e., p>0.05) compared to the PBS-treated control group by unpaired, two-tailed Student’s t test. (B and D) Data represent mean ± SEM and are representative of at least two independent experiments with n = 3 per experiment. The vertical dotted lines indicate the EC₅₀ values of the dose-response curves. The numbers on top of the black arrows indicate the fold difference between the EC₅₀s.