Fig. 2.

Trehalose did not inhibit autophagic flux but accelerated p62 turnover. (A–C) Hepa1-6 cells were treated for 24 h with 50 or 100 mM trehalose, with or without chloroquine (CQ), while untreated cells were used as controls. Total cell lysates were analyzed by western blotting using anti-p62, LC3 and β-actin antibodies (A) and bands were quantified (B, C). β-Actin was used as a loading control. Representative images and quantitative data (n = 4) are shown. Values are means ± SD. Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01. (D) Hepa1-6/RFP-GFP-LC3 cells were treated with 50 mM trehalose or 10 μM CQ for 8 h (upper panel). Hepa1-6 cells were treated with 50 mM trehalose or 10 μM CQ for 8 h and stained with LysoTracker Red for 30 min (lower panels). Images were acquired by confocal fluorescence laser microscopy. Scale bars are 20 µm and 50 µm, respectively. (E, F) Hepa1-6 shLuc (control)/shAtg5 cells were pretreated with 50 mM trehalose or 10 μM CQ for 24 h and then treated with 200 μM cycloheximide (CHX) for the indicated times. Total cell lysates were analyzed by western blotting using anti-p62 and LaminB1 antibodies and quantified. LaminB1 was used as a loading control. Representative images are shown (E). The quantitative data (n = 3) is shown as relative to the values at time 0 min for each cell type and under each experimental condition (F). Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01.