Figure 4.

Exogenous ceramide fails to rescue CD4⁺ T cells from ASM inhibition. (a–c) Naive CD4⁺ T cells were stimulated with anti-CD3/CD28 and treated with various concentrations of exogenous C6-ceramide (Cera 1: 10 nM; Cera 2: 100 nM; Cera 3: 1000 nM), followed by FACS (fluorescence-activated cell sorting) analysis of T-cell activation (a) and CD25 expression (b) at 48 h, and western blotting of intracellular CD3/CD28 signaling components induced by 1000 nM C6-ceramide at the indicated time (c). (d) CFSE-labeled naive CD4⁺ T cells were stimulated with anti-CD3/28 antibody-coated beads for 72 h in the presence of different doses of C6-ceramide as indicated above together with or without imipramine (10 μM), followed by FACS detection of CFSE and CD25 expression. (e) Anti-CD3/CD28 antibody-coated beads of activated CD4⁺ T cells were treated with imipramine (10 μM) in combination with various doses of C6-ceramide as above, and CD3 dynamics was visualized at 60 min after anti-CD3/CD28 stimulation by confocal microscopy. Data are representative of three to five independent experiments