Fig. 3

A ATG7-knockdown inhibits NICD degradation. HeLa and H1299 cells were transiently transfected with ATG7 shRNA or control shRNA using Lipofectamine 3000 and incubated for 24 h. Cells were then starved in HBSS for 4 h. Total cell proteins (30 μg) were separated by 10% or 12% SDS-PAGE and analyzed by Western blotting using antibodies against LC3, SQSTM1, ATG7, and SNAI1. \(\beta \)-actin was used as the loading control. NICD levels in HeLa cells (B) and H1299 cells (C) were quantified using NIH ImageJ software. (D) NICD degradation is independent of the SQSTM1-associated proteasome. HeLa cells were transfected with control or SQSTM1 siRNA using Lipofectamine 3000 and incubated for 24 h. Then, cells were starved in HBSS for 4 h. Total proteins were subjected to Western blotting. Relative levels of NICD (E) and SNAI1 (F) were quantified. G NICD degradation is independent of the proteasomal system. HeLa cells were starved in the presence of either 20 μM chloroquine (CQ) or 20 μM MG-132, and relative levels of NICD (H) and SNAI1 (I) were quantified. Data represent the mean (± S.D.) of three independent experiments (**P < 0.01)