Fig. 7. SPTLC1 deficiency increases intracellular ROS by enhancing NOX activity.

(A) WT and KO naïve CD4⁺ T cells were differentiated under T_H17 conditions for 12 hours and mitochondrial ROS measured using Mitosox red. The left panel is a representative histogram, and the right panel shows the cumulative data (MFI) from n = 3 biologically independent samples. (B) WT and KO naïve CD4⁺ T cells were differentiated under T_H17 conditions for 4 days with or without DPI (10 nM) and scored for intracellular cytokines. The left panel is a representative flow figure, and the right panel shows the cumulative data from n = 4 biologically independent samples. (C) WT and KO naïve CD4⁺ T cells cross-linked with anti-CD3 for 15 min with DCFDA and read in flow cytometry. The left panel is a representative histogram, and the right panel shows the cumulative data (MFI) from n = 3 biologically independent samples. (D) WT and KO naïve CD4⁺ T cells cross-linked as in (C) and membrane fraction immunoblotted for p47 ^phox. Each lane is a pooled sample from four to eight mice. The left panel shows representative blot, and the right panel is the quantitative data from three independent experiments. (E) CD4⁺ T cells cross-linked as in (C) and stained for gp91^phox (green) and p47 ^phox (red) and analyzed using confocal microscopy. The left panel shows the representative confocal picture, and the right panel shows the number of colocalization spots per cell. n = 3 biologically independent samples. (F) WT and KO Naïve CD4⁺ T cells were nucleofected with control or p47^phox siRNA, differentiated into T_H17 cells, and scored for intracellular cytokines. The left panel is a representative flow figure, and the right panel shows the cumulative data. Each dot represents cells pooled from three mice. n = 3 independent experiment. All data presented as means ± SEM: *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant.