Expression of the cell death markers activated caspase-3 and LC3 I/II in TUBO cells. Expression of the cell death markers activated caspase-3 and LC3 I/II in TUBO cells. A, TUBO cells were treated with 62.5 nm staurosporine for 16 hours to induce apoptosis. B, TUBO cells were serum-starved and treated with rapamycin (200 nmol/L) or bafilomycin (50 nmol/L) for 24 hours to induce autophagy. C and D, cells were infected with KM100 (MOI 10) for 1 hour and medium was added with or without 5 μmol/L MTX. Total protein was harvested at the indicated times posttreatment for Western blot analyses to detect the levels of activated caspase-3 and LC3-I and LC3-II. The blots were used to calculate the ratio of LC3-II:LC3-I by densitometry using ImageJ software. The experiments were repeated more than three times and a representative blot is shown. E, TUBO cells treated as described earlier were used to collect supernatants after 24 hours of treatment to measure the level of HMGB1. The level of HMGB1 plotted is from pooled samples of three independent experiments. Samuel T. Workenhe et al. Cancer Immunol Res 2013;1:309-319 ©2013 by American Association for Cancer Research