Figure 2. Autophagic activity in peripheral T cells from lupus-prone mice is raised compared with controls T cells sorted from spleens obtained from 8–12 week-old control CBA/J and lupus MRLlpr/lpr mice (A) or from 12–20 week-old control BALB/c and lupus NZB/W mice (B) were left unstimulated (steady-state) or stimulated by 50 ng/mL PMA and 1 µM Ionomycin (PMA/Iono) at 37°C for 16h. When indicated, cells were treated (+) or not (-) during the last 4 h of the culture with 5 µg/mL pepstatin A and 5 µg/mL E64d to block lysosomal degradation. Cell lysates were resolved by SDS-PAGE, transferred onto PVDF membranes before staining with anti-LC3 Ab. Loading controls were performed by staining actin-β chain. Each immunoblot is representative of at least five independent experiments with identical results. *Band corresponding to the Ig heavy chain retained in lysates obtained from oldest lupus mice. LC3-II/β-actin band intensity ratios are indicated as numbers under each immunoblot. (C and D) LC3-II levels were evaluated by densitometry and normalized to β-actin band intensities for at least five independent experiments. Histogram bars represent the means of individual experiments with standard errors. *p < 0.05, **p < 0.01, ***p < 0.001 using paired t-test between control and lupus conditions. Activation of sorted T cells from control and lupus mice was assessed by flow cytometry with CD69 staining (E). Dotted black lines and solid gray lines represent respectively control and lupus mice.