03 gp62 c

Tissues were snap-frozen in liquid nitrogen, ground using Cryomill in liquid nitrogen at 25 Hz for 2 min, and then lysed in Tris lysis buffer (1M Tris-HCl, 1M NaCl, 0.1M EDTA, 10% NP40). Protein concentrations were measured using the Bio-Rad BCA reagent. Samples were probed with antibodies against Atg7 (Sigma Aldrich, A2856, RRID:AB_1078239), Lkb1 (Santa Cruz Biotechnology, sc-32245, RRID:AB_627890), LC3 (Novus Biologicals, NB600-1384, RRID:AB_669581), p62 (American Research Products, 03-GP62-C, RRID:AB_1542690), p-ACC^S79 (Cell Signaling, 3661, RRID:AB_330337), ACC (Cell Signaling, 3676, RRID:AB_2219397), Atg5 (Abcam, ab108327, RRID:AB_2650499), p-S6^S235/236 (Cell Signaling, 4858, RRID:AB_916156), S6 (Cell Signaling, 2217, RRID:AB_331355), and β-actin (Sigma Aldrich, A1978, RRID:AB_476692). Western blots were quantified with Image J (National Institutes of Health). The intensities of bands were used to calculate relative ratios of the indicated protein over loading control (β-actin), which were then normalized based on the corresponding ratio in the wild type control sample.

Protein lysates from tumor cells treated with Ivermectin were prepared using RIPA buffer. Activation and cleavage of caspases was evaluated using antibodies specific for caspase-1 (NOVUS, NBP1–45433) and caspase-3 (Cell Signaling, 9665). Induction of autophagy was validated using antibodies specific for p62/SQSTM1 (American Res., 03-GP62-C) and LC3 (Cell Signaling, 4599).