Figure 8.

Inhibition of LAT1 expression in stimulated T lymphocytes suppresses activation-induced citrulline transport. Primary human CD3⁺ T lymphocytes were isolated from blood of healthy donors by negative selection. CD3⁺ T cells were electroporated in the absence of RNA (−) or transfected with non-target RNA (nt) or LAT1 siRNA (siLAT1). After 4 h, cells were stimulated with anti-CD3/anti-CD28-coupled beads for 24 h. Non-stimulated T cells were included as further controls (Co). (A,B) LAT1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression was determined by Western blot; (A) representative blot, (B) quantification of three independent experiments. Data are expressed as percentage of the respective LAT1/GAPDH ratio obtained in the same experiment for cells stimulated for 24 h without any transfection (mean ± SD, n = 3). (C) The uptake of 20 µM [¹⁴C]citrulline over 30 s was evaluated and expressed as percentage of values obtained with 24 h-stimulated, non-transfected cells (19.56 ± 9.49 pmol citrulline/10⁶ cells, mean ± SD, n = 8–12 from 3 to 5 donors). Statistical analyses were performed using one-way analysis of variance with Tukey’s post hoc test (*** or ^###p < 0.001, ** or ^##p < 0.01, n.s.: p ≥ 0.05).