Fig 1: The affinity and geometry of the design agonists bias intracellular signaling and primary stem cell differentiation(A and B) Representative western blot images of intracellular levels of phospho-STAT3 (Tyr705) (A) and phospho-STAT5 (Tyr694) (B) proteins after treatment of NFS-60 cells with 1 nM of different designs (saturating conditions) for 5 min (left) or 30 min (right). Total protein staining was used as a loading control. Five biological replicates of this experiment showed the same trend of higher pSTAT3/pSTAT5 ratio by the design agonists (see Figure S21). (C and D) The pSTAT3 (C) and pSTAT5 (D) levels were quantified after 5 and 30 min from those replicates. Shown is the level of pSTAT3 or pSTAT5 divided by the total protein-normalized STAT3 or STAT5 levels, respectively. (E and F) To probe the effect of the design agonists (100 ng/mL) and rhG-CSF (10 ng/mL) on healthy donors’ CD34+ HSPCs, proliferation assays were performed without (E) or with (F) the addition of 50 ng/mL SCF and 20 ng/mL IL-3. Ori0 without SCF and IL-3 were tested at three concentrations: 1 ng/mL (triangle, left), 10 ng/mL (triangle, right), and 100 ng/mL (diamond). Shown are the mean (points) and standard deviation (shades) of three parallel replicates. (G–I) Additionally, we performed CFU assays of healthy donors’ CD34+ and progenitors incubated on semi-solid medium supplemented with corresponding cytokines and design agonists. IL-3 and SCF were added to all samples. Shown is the fold change to rhG-CSF of the quantified CFUs of granulocytes (CFU-G) (G), granulocyte-macrophages (CFU-GM) (H), and macrophages (CFU-M) (I). The circles represent the obtained values for each condition of three independent experiments with two parallel replicates each. The indicated p values were estimated by an ordinary one-way ANOVA followed by a Tukey HSD test.