Figure 3

Proliferation of sorted CD4⁺CD25⁺Tregs. (A) Overexpression of FR4 or FR4D3 protein in sorted CD4⁺CD25⁺ Tregs. Wt, wild type cells; Over-FR4, overexpression of FR4; Over-FR4D3, overexpression of FR4D3. Each column represents the intensity abundance ratio of the FR4 or FR4D3 band versus the actin band in each lane. Panels show a representative of three independent experiments. Error bars represent the standard deviation from three independent experiments. *, P < 0.05, vs. wild type cells. (B) FA dose-response of Tregs’ proliferation. The CD4⁺CD25⁺ Tregs were stimulated with various concentrations of FA (0, 2, 4, 6, 8, 10 ng/mL) in the presence of IL-2, anti-CD3/anti-CD28 (pre-coated and soluble, respectively). Panels show a representative of three independent experiments. Error bars represent the standard deviation from triplicate wells.*, P < 0.05 vs. untreated cells. (C) The FR4 or FR4D3 CDS was cloned directly into the pCI-neo vector and transiently transfected into FACS-sorted CD4⁺CD25⁺ Tregs, followed by stimulation with or without combinations of FA, IL-2, and anti-CD3/anti-CD28 (pre-coated and soluble, respectively). Wt represents no overexpression of either FR4 or FR4D3; FR4 represents the overexpression of FR4; FR4D3 represents the overexpression of FR4D3. Panels show a representative of three independent experiments. Error bars represent the standard deviation from triplicate wells. *, P < 0.05.