Figure 2.

OA-challenge inhibits activation-induced macroautophagy activation. Mouse T_H1 cells were incubated in the presence of different concentrations of OA for 48 h. Cells were then either left resting (Rest) or 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) immunoblot (LC3-II degradation) or (b) immunofluorescence (LC3⁺ vesicle turnover). ACTB was used as loading control for normalization of values. Bar graphs represent mean+SEM of autophagy flux, measured as the difference between the intensity of the LC3-II band in cells activated in the presence or absence of N/L from 5 independent experiments or as the difference between the number of LC3⁺ puncta in cells cultured in the presence or absence of N/L from 10 fields per sample containing 30 cells per field (*P < 0.05. ANOVA). (c) Murine T_H1 cells were incubated for 48 h in the presence or absence of different concentrations of OA and then activated with plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. N/L were added for the last 1, 2 or 3 h of the 24-h activation period. Bar graphs represent mean+SEM of autophagy flux from 3 independent experiments, measured as the difference between the intensity of the LC3-II band in cells cultured in the presence or absence of N/L for different periods of time or the difference between LC3-II levels in cells incubated for 1 or 3 h with N/L (*P < 0.05; ANOVA).