Fig. 7: KPNB1 inhibition-induced autophagy eliminates TRAIL-induced cleaved caspase-8.

a U87 and U251 cells were pretreated with CQ (40 μM) or Baf-A1 (5 nM) along with IPZ (16 μM) or IVM (16 μM) for 24 h and further with TRAIL (U87, 50 ng/ml; U251, 100 ng/ml) for 3 h. P62 in cell lysates was immunoprecipitated followed by western blot. b Representative images showed colocalization of p62, caspase-8 p43/p41/p18, and LC3 in U251 cells when treated with Baf-A1 (5 nM) and IPZ (16 μM) for 24 h and further with TRAIL (100 ng/ml) and z-DEVD-FMK (50 μM) for 8 h. Magnification, × 60; scale bar 25 μm. c U87 and U251 cells expressing shLC3B or not were treated with IPZ (16 μM) and CQ (40 μM) or Baf-A1 (5 nM) for 24 h and further with TRAIL (U87, 50 ng/ml; U251, 100 ng/ml) for 2 h. Then, cells were subjected to CHX pulse-chase assay by treating with CHX (U87, 20 μg/ml; U251 100 μg/ml) and z-VAD-FMK (20 μM) for indicated period of time. Representative images of western blot were shown in the upper panel. Quantification of grayscale ratio of cleaved caspase-8/α-tubulin by Photoshop software were shown in the lower panel. Results represent mean ± SD from two independent experiments. d, e U251 cells were pretreated as in a and further with z-IETD-FMK (20 μM) and TRAIL (100 ng/ml) for 6 h (western blot) or 24 h (flow cytometry), then subjected to flow cytometry (d) and western blot (e). Results represent mean ± SD from three independent experiments. GAPDH and α-tubulin was used as loading controls. *P < 0.05