Fig. 4: Climacostol impairs autophagic flux.
From: Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells
B16-F10 cells were cultured with 30 μg/ml climacostol (CLIMA) or control vehicle (CTRL) for increasing times. Chloroquine (CQ; 10 μM, 6 h) was also used. a Confocal fluorescence imaging of cells transiently transfected with mRFP-GFP-LC3 plasmid. This tandem fluorescent-tagged LC3 reporter results in both green (GFP) and red (RFP) fluorescence: autophagosomes appear yellow (with green and red fluorescence) and autolysosomes as only red vacuoles. Scale bar: 30 μm. Inserts represent enlarged image details highlighting the presence of red (arrows) and yellow puncta. b The number of autophagosomes and the number of autolysosomes in the merged images were counted and the total number of puncta per cell was calculated as percentage (>30 cells per experimental condition). *p < 0.05 relative to CTRL. c Western blotting images of LC3 and p62 expression. LDH was used as internal standard. Images and data are representative of 3 independent experiments
