Figure 7
From: Piperlongumine induces autophagy by targeting p38 signaling
p38 signaling is required for autophagy induction by PL. (a–c) U2OS/GFP-LC3 cells were transfected with pcDNA3 (Ctrl) or pcDNA3/p38-DN constructs and selected with 200 μg/ml hygromycin for 2 weeks for the establishment of stable expression cell lines. (a) The control and p38-DN stable expression cells were treated with 10 μM PL for 16 h. After the treatment, the WCE was isolated for western blot detection of p38 signaling targets, anti-oxidant enzymes and autophagy marker (LC3). Thirty micrograms of total proteins were loaded. GAPDH served as a loading control. (b) The control and p38-DN stable expression cells were treated with 10 μM PL for 16 h. GFP-LC3 fluorescence was captured with fluorescence microscopy after the treatment. White arrows indicate punctate GFP-LC3 fluorescence (autolysosomes/autophagosomes). (c) The control and p38-DN stable expression cells were treated with 10 μM PL for 48 h. The cell viability was determined with MTT assay after the treatment. Data are shown as mean±S.D. (n=4). *P<0.05. (d) U2OS/GFP-LC3 cells were pre-treated with 10 μM SB 203580, a p38 inhibitor, or 20 μM PD 98059, a p44/42 inhibitor for 6 h, followed by treatment with 10 μM PL for 16 h. GFP-LC3 fluorescence was captured with fluorescence microscopy after the treatment. White arrows indicate punctate GFP-LC3 fluorescence (autolysosomes/autophagosomes). (e) U2OS/GFP-LC3 cells were pre-treated with 10 μM SB 203580, a p38 inhibitor or 20 μM PD 98059, a p44/42 inhibitor for 6 h, followed by treatment with 10 μM PL for 48 h. The cell viability was determined with MTT assay after the treatments. Data are shown as mean±S.D. (n=4). *P<0.05. (f) Role of PL in autophagy. Schematic diagram depicts mechanism of action of PL to induce autophagy. PL increases intracellular ROS levels, thereby activating p38 signaling and enhancing cell autophagy
