Fig. 1

Autophagy activation stimulates NICD degradation in cancer cells. A HeLa and H1299 cells were treated with 100 nM rapamycin (Rapa) or starved in HBSS medium for 4 h in the presence or absence of 20 μM chloroquine (CQ). Total cell extracts (30 μg) were separated by 10% SDS-PAGE and analyzed by Western blotting using primary antibodies against LC3, SQSTM1, p-ULK1-S555, p-ULK1-S757, p-AMPK-T172, AMPK, p-mTOR-S2248, mTOR, SNAI1, NICD, and NOTCH1. \(\beta \)-actin was used as the loading control. B, C Quantification of NICD cellular levels in cancer cells. The relative protein levels of NICD in HeLa (B) and H1299 (C) cells, and phosphorylation of ULK at S555 (D), AMPK at T172 (E) and mTOR at S2448 (F) were quantified using NIH ImageJ software. Data represent the mean (± S.D.) of three independent experiments (*P < 0.05 and **P < 0.01). (G) Co-immunoprecipitation. HeLa cell extracts were immunoprecipitated using anti-LC3 (left) or anti-NICD (right) antibodies. The bound proteins were analyzed by Western blotting using the indicated antibody. Whole cell extracts (5% input) were also assessed to control for the amount of total protein. IgG indicates the nonspecific mouse antibody that was used as the negative control. H Co-immunoprecipitation in starved cells. Total proteins extracted from starved HeLa cells were immunoprecipitated using anti-LC3 antibodies, and bound proteins were analyzed as described above. I Immunocytochemistry. HeLa cells were cultured on coverslips for 24 h and then starved in HBSS for 4 h. After fixing the cells with paraformaldehyde, the cells were incubated for 24 h at 4 \(^\circ \)C with two antibodies: LC3 and NICD (top) or SQSTM1 and NICD (bottom). The cells were washed and then incubated with secondary antibodies (FITC-conjugated anti-mouse antibody or TRITC-conjugated anti-rabbit antibody). The coverslips were mounted onto glass slides using a mounting medium containing DAPI, and all images were captured by fluorescence microscopy. Protein colocalization was quantified using NIH Image J software (plugin). Scale bar, 10 μm. Data represent the mean (± S.D.) of three independent experiments (**P < 0.01). J, K In vitro interactions between SNAI1 and LC3, SNAI1 and SQSTM1, NICD and LC3, and NICD and SQSTM1. GST-NICD (J) or GST-SNAI1 (K) proteins were immobilized on glutathione beads and incubated with purified LC3 (left) or SQSTM1 (right) for 1 h at 4 \(^\circ \)C. After washing, bound proteins were subjected to 10% SDS-PAGE and visualized by Western blot using anti-LC3, anti-SQSTM1, anti-SNAI1, or anti-NICD