Figure 5.

HFD-fed mice show reduced TCR-induced autophagy activation. CD4⁺ T cells isolated from HFD or control diet (CD) fed mice were either left resting (Rest) stimulated (Activ) with plate bound anti-CD3 and anti-CD28 antibodies for 24 h. NH₄Cl and leupeptin (N/L) were added for the last 3 h of the 24-h stimulation to assess autophagy flux measured by (a) immunofluorescence (LC3⁺ vesicle turnover) or (b) immunoblot (LC3-II degradation). ACTB was used as loading control for normalization of values. Bar graphs represent mean+SEM of autophagy flux, measured as the difference between the number of LC3⁺ puncta in cells cultured in the presence or absence of N/L from 5 independent experiments or the difference between the intensity of the LC3-II band in cells activated in the presence or absence of N/L from 4 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. ANOVA or two-tailed t-test, respectively).