Fig. 6

Correlation between autophagy activity and the NICD level in cancer tissues and the effect of autophagy on cancer cell migration. A Total lysates from cancer tissues from cervical (left) and lung (right) cancer patients were prepared. Total protein (30 μg) from each sample (C, cancer tissue; N, normal tissue) was separated by 10% and 12% SDS-PAGE and analyzed by Western blotting using primary antibodies against LC3 ATG5, ATG12, ATG7 p-ULK1-S555, p-ULK1-S757, p-AMPK-T172, SNAI1, and NICD. \(\beta \)-actin was used as the loading control. Expression levels of autophagy proteins (p-ULK1-S555, p-ULK1-S757, p-AMPK-T172) and EMT mediators (NICD, SNAI1) in cervical (B) and lung (C) cancer tissues were quantified using NIH ImageJ software. Data represent the mean (± S.D.) of three independent experiments (**P < 0.01) (D) Migration assay. Confluent HeLa cells were pre-treated under various conditions (control, rapamycin, Bafilomycin A1, starvation in HBSS, and combinations of these conditions) for 4 h, and then the confluent layer was scratched using a blue tip. Cells were continuously incubated under the same conditions for 24, 48, or 72 h. At each timepoint, cell migration images (50× magnification) were captured by microscopy. Wound closures (percentage of the average migratory distance of cells relative to the original wound edge) were quantified E. Data represent the mean (± S.D.) of at least three independent experiments (**P < 0.01). F Matrigel invasion assay. HeLa cells were transiently transfected with pcDNA, FLAG-ATG7, or CRISPR-Cas9-ATG7 plasmids using Lipofectamine 3000 and incubated for 24 h. After invasion of the transwell insert, cells were starved in HBSS for 4 h and then stained with propidium iodide. Cell images (F) were captured by microscopy (10× objective magnification) and quantified from 10 randomly selected fields (G). Data represent the mean (± S.D.) of three independent experiments (**P < 0.01). H A schematic representation of autophagy-dependent NICD degradation during cancer progression. Autophagy signals lead to association of NICD and SNAI1 with LC3 and SQSTM1 to sequester complexes into autophagosomes for degradation. Consequently, nuclear translocation of NICD and SNAI1 is decreased causing transcriptional suppression of EMT-related genes (N-cadherin, Zeb1, Vimentin) and transcriptional activation of E-cadherin. As a result, autophagy-dependent degradation of NICD and SNAI1 plays a critical role in the regulation of cancer progression