Figure 5. LA induces T cell death through promoting mitochondrial ROS production and lipid peroxidation.

(A-C) Splenic T cells purified from normal C57BL/6 mice were treated with 400μM LA, OA or BSA for 20 hours. Mitochondrial ROS production in CD8⁺ and CD4⁺ T cells were analyzed by MitoSox staining (n=8). Average percentages of Mitosox⁺ CD8⁺ and MitoSox⁺ CD4⁺ T cells in each group are shown in panel B and C, respectively.

(D) Confocal microscopy analysis of Mitospy™ NIR DiIC1 (top) and PMP70 (70-kDA peroxisomal membrane protein) (bottom) in sorted T cells treated with BSA, LA or OA for 30 mins.

(E, F) Mitochondrial potential in CD8⁺ (E) or CD4⁺ (F) T cells was measured by flow cytometry with Mitospy™ NIR DiIC1 staining.

(G-I) Splenic T cells were treated with BSA, LA or OA (400μM) for 4 hours. Representative histogram of anti-4-HNE (4-hydroxynonenal) lipid peroxidation staining in CD8⁺ or CD4⁺ T cells was shown in panel G. Mean fluorescent intensity (MFI) of anti-4-HNE in CD8⁺ and CD4⁺ T cells is shown in panel H and I, respectively.

(J) Real-time PCR analysis of Cpt1b gene expression in sorted T cells treated with BSA, LA, or OA (400μM) for 12 hours.

(K, L) Analysis of mitochondrial ROS production by Mitosox staining in CD8⁺ (K) or CD4⁺ (L) T cells treated with LA (400μM) in the presence or absence of etomoxir (1μM), a specific Cpt1 inhibitor, for 20 hours.

(M, N) Analysis of Annexin-V⁺ CD8⁺ (M) or CD4⁺ (N) T cells treated with LA (400μM) in the presence or absence of 1μM etomoxir for 20 hours.

Results represent 3 independent experiments. Data are shown as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Also see Figure S4.