Fig. 3.

KPNB1 inhibition enhances TRAIL sensitivity by promoting ATF4-mediated DR5 expression. a, b shKPNB1-expressing (a) or IPZ (16 μM)-treated (b) U87 cells were treated with pan-caspase inhibitor Z-VAD-FMK (20 μM) for 5 h to block caspase-8 cleavage and further treated with TRAIL (100 ng/ml) for 1 h. DISC in cell lysates was immunoprecipitated using caspase-8 antibody followed by western blot. c U87 cells expressing shKPNB1s and/or shDR5 were treated with TRAIL (50 ng/ml, 6 h, for western blot; 30 ng/ml, 24 h, for flow cytometry, the same below unless noted) and subjected to western blot or flow cytometry. Results represent mean ± SD from three independent experiments. *P < 0.05. d U87 cells expressing shDR5 were treated with IPZ (16 μM) for 24 h and further treated with TRAIL, then subjected to western blot or flow cytometry. Results represent mean ± SD from four independent experiments. *P < 0.05. e, f U87 cells either expressing shKPNB1s (e) or pretreated with IPZ (16 μM) for 24 h (f) were treated with antagonistic antibody to DR4 or DR5 or IgG1 isotype control antibody (all 5 μl/ml) for 1 h and further with TRAIL for 24 h, then subjected to flow cytometry. Results represent mean ± SD from three independent experiments. *P < 0.05. g Western blot analysis of indicated proteins in shKPNB1-expressing or IPZ (16 μM)-treated U87 cells. h, i Real-time PCR analysis of indicated genes in U87 cells expressing shKPNB1s and/or shATF4 (h), and in U87 cells expressing shATF4 and/or treated with IPZ (16 μM) i Results represent mean ± SD from three independent experiments. *P < 0.05. j, k Western blot analysis of indicated proteins in U87 cells treated as in h and i. GAPDH was used as the loading control