Fig. 2

Impaired CD4⁺ T cell proliferation is associated with increased GSK3 activity. a Immunoblot analysis of c-Myc expression in total lysates of CD4⁺ T cells from WT and Gimap5^sph/sph mice stimulated with αCD3/αCD28 or during resting conditions. b Myc mRNA levels in resting and αCD3/αCD28-activated (24 h) CD4⁺ T cells. Data represent mean expression ± SD relative to unstimulated WT cells (n = 6). c Phosphorylation c-Myc (T58) after 24 h of αCD3/αCD28 stimulation. Proteasomal inhibitor MG132 was added after 20 h of stimulation. Ratio of p-c-Myc (T58) to total c-Myc in MG132-treated Gimap5^sph/sph CD4⁺ T cells relative to WT (n = 5). d C-Myc expression in WT and Gimap5^sph/sph CD4⁺ T cells stimulated for 24 h with αCD3/αCD28 ± 2.5 mM LiCl. e Proliferation of WT and Gimap5^sph/sph CD4⁺ T cells in the presence/absence of αCD3/αCD28 and/or GSK3 inhibitors BIO (100 nM) or LiCl (2.5 mM) as measured by CFSE dilution after 3 days. Experiments were repeated five times and representative plots from a single experiment are shown. f Representative images of NFATc1 localization in resting and αCD3/αCD28-activated WT and Gimap5^sph/sph CD4⁺ T cells. Bright detail similarity quantification of NFATc1 nuclear localization upon stimulation (g) and in the presence/absence of BIO (h) for 4 h (n = 6). Graphs depict mean values ± SEM. ImageStream data represent average values of >500 CD4⁺ T cells per sample; all experiments were performed at least three times. Statistical significance is determined by Student’s two-tailed test. BF bright field