Analysis for molecular markers of autophagy and apoptosis in C225-NP-treated cancer cells.

(a) Cellular proteins were lysed at the indicated time after treatment with IgG-NP particles or C225-NP (0.6×1010 particles) and subjected to Western blotting. In HCC827 but not in H520 cells, increased PARP cleavage and LC3-II was observed at 48 h and 72 h after treatment with C225-NP and when compared with IgG-NP treatment and untreated control. The intensities of the amount of LC3-II bands were semi-quantified by ImageJ software (National Institutes of Health, Bethesda, MD) (b) HCC827 cells were treated with IgG-NP or C225-NP for 68 hrs, cells were further cultured with or without protease inhibitors [10 µg/ml E-64-d and 10 µg/ml pepstatin A (BIOMOL International L.P., Plymouth Meeting, PA)] for 4 hrs. Cellular proteins were lysed and immunoblotted with anti-LC3 antibody. LC3-II protein levels were increased in the presence of protease inhibitors in all of the groups indicating occurrence of autophagy. The intensities of the amount of LC3-II bands were semi-quantified by ImageJ software (National Institutes of Health).