Supplementary Fig. 4: PlGF-induced increase in IL-17 production and p-STAT3 activation via FLT1 and NRP1 receptor.

(a) Downregulation of PlGF expression in CD4⁺ T cells by siRNA. CD4⁺ T cells were transfected with siRNAs for Flt1 or Nrp1 using a T-cell nucleofector kit. After 24 hours, expressions of Flt1 and Nrp1 mRNA were determined by real-time PCR. * P < 0.05 compared to control siRNA (Con). (b) Sorted CD4⁺ T cells (n = 16) were cultured in RPMI 1640 with 10% FBS and stimulated with PlGF_CM (100 ng/ml) for 72 hours in the absence or presence of soluble FLT1 (sFLT1, 10 μg/ml) and soluble NRP1 (sNRP1, 10 μg/ml). Levels of IL-17 in culture supernatants were measured by ELISA. * P < 0.05 compared to cells incubated with PlGF_CM but not sFLT1 or sNRP1. Data presented in (a), and (b) are the mean and SEM of more than three independent experiments. (c) CD4⁺ T cells were incubated with Con_CM or PlGF_CM (100 ng/ml) for the indicated times. Phosphorylated STAT3 expression was measured using flow cytometry. Presented data are from two independent experiments with similar results.