Figure 6
From: Piperlongumine induces autophagy by targeting p38 signaling
PL activates p38 signaling. (a) U2OS/GFP-LC3 cells were pre-treated with 5 mM NAC for 6 h, followed by treatment with 2.5 μM PL for 16 h. The WCE was isolated after the treatment. Thirty micrograms of total proteins were loaded for western blot analysis. GAPDH served as a loading control. (b and c) U2OS cells were transfected with pcDNA3 (Ctrl), pcDNA3/p38-WT and pcDNA3/p38-CA and selected with 200 μg/ml hygromycin for 2 weeks for the establishment of stable expression cell lines. (b) The control and p38 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. (c) The control and p38 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<0.01. (d) U2OS/GFP-LC3 cells were transfected with pcDNA3 (Ctrl), pcDNA3/p38-WT or pcDNA3/p38-CA for 48 h. The cells were then exposed to 10 μM PL for 16 h. GFP-LC3 fluorescence was captured with fluorescence microscopy. White arrows indicate punctate GFP-LC3 fluorescence (autolysosomes/autophagosomes)
