Fig. 3: Climacostol impairs autophagic flux.
From: Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells
a, b Western blotting images of LC3 and p62 expression in B16-F10 cells cultured with 30 μg/ml climacostol (CLIMA) or control vehicle (CTRL) for 24 h, both in the absence and presence of chloroquine (CQ; 10 μM, 6 h). LDH was used as internal standard. Lower panels: densitometric analysis of LC3-II and p62 relative to their respective standard. Results are expressed as fold change of CTRL. Images and data are representative of 3–5 independent experiments. *p < 0.05, **p < 0.005 and ***p < 0.0001 relative to CTRL. c Electron microscopy images presenting ultrastructure of B16-F10 cells cultured with 30 μg/ml CLIMA or CTRL for 6 h. The panels 1–3 depict representative control cells at increasing magnifications: (1) whole cells; (2) an early or initial autophagic vacuole (Avi), containing morphologically intact ribosomes. The electron-lucent cleft between the two limiting membranes is visible. A dense lysosome (Ly) is also found in contact with the outer limiting membrane of the autophagosome and a normal mitochondria (mi); (3) a late or degradative autophagic vacuole (Avd) containing partially degraded cytoplasmic material. The panels 4–7 depict representative climacostol-treated cells at increasing magnifications: (4) whole cells showing abundant black melanosomes; (5) note the presence of numerous autophagosome-like compartments in the cytoplasm; (6) higher magnification of melanosomes and (7) mitochondria with swollen cristae. Scale bars: 1 and 4: 2 μm; 2 and 3: 200 nm; 5, 6 and 7: 500 nm. Images are representative of 3 independent experiments
