Neddylation improves CD4⁺ T cell survival via a mitochondria-dependent mechanism.

(A) Representative dot plots showing Annexin V and 7AAD staining of splenic CD4+ T cells from Uba3fl/fl and Uba3ΔT mice at days 0, 7 and 11 p.i., bar graphs showing the proportions of total apoptotic Annexin V+ cells in the gated CD4+ T cell population (n = 6 per group). (B) Representative histograms showing Fas and FasL expression on splenic CD4+ T cells from Uba3fl/fl (black line) and Uba3ΔT mice (gray-filled area) at days 7, 11 p.i., data represent 3 mice per group from three independent experiments. (C) Left, representative immunoblots of Bcl-2, Bcl-XL and Bim in splenic CD4+ T cells from Uba3fl/fl and Uba3ΔT mice at days 0,5,10 p.i., numbers are density of the bands quantified by scanning densitometry, normalized to GAPDH, relative to that of uninfected Uba3fl/fl mice. Right, bar graphs showing fold densitometry of indicated molecules in Uba3ΔT CD4+ T cells compared to that in Uba3fl/fl counterparts at the same time point (n = 3–4 per group). (D) Left, flow cytometry analysis of mitochondrial membrane potential (ΔΨm, TMRE staining) in splenic CD4+ T cells from Uba3fl/fl (red) and Uba3ΔT mice (blue) at days 0,7,11 p.i., cells pretreated with FCCP (+FCCP, black and gray dashed line) to eliminate TMRE signal served as negative controls. Right, bar graphs showing loss of ΔΨm in gated splenic CD4+ T cells from naïve and P. yoelii 17XNL-infected mice (n = 4 per group). (E) Bcl-2 expression in CD4+ T cells from Uba3fl/fl and Uba3ΔT mice stimulated with CD3/CD28 Dynabeads for 0, 48 and 60 h. (F) Representative dot plots showing Annexin V and PI staining of CD4+ T cells from Uba3fl/fl and Uba3ΔT mice, either prior to (0 h) or after stimulation with CD3/CD28 Dynabeads in the presence of 300 nM ABT737 (+) or DMSO (–) for 60 h. Data are presented as mean±SEM from three or more independent experiments. *pppt test.