Fig. 5: KPNB1 inhibition cripples cap-dependent translation of FLIP and TRAIL tolerance by ATF4/4E-BP1 axis.

a U87 cells expressing shKPNB1s and FLIP_L or FLIP_S were treated with TRAIL and subjected to western blot or flow cytometry. Results represent mean ± SD from three independent experiments. b U87 cells expressing FLIP_L or FLIP_S were treated with IPZ (16 μM) for 24 h and further with TRAIL, then subjected to western blot and flow cytometry. Results represent mean ± SD from three independent experiments. c U87 cells expressing shKPNB1s or treated with IPZ (16 μM) for 24 h were subjected to cap-binding assay. d Protein synthesis was measured by OPP incorporation after treating U87 and U251 cells with 16 μM IPZ or IVM for 24 h, or with 100 μg/ml CHX for 4 h as a negative control for protein synthesis Results represent mean ± SD from one of the three independent experiments in triplicates. e U87 cells expressing shKPNB1s and/or sh4E-BP1 were subjected to cap-binding assay. f U87 cells expressing sh4E-BP1 were treated with IPZ (16 μM) for 24 h and subjected to cap-binding assay. g U87 cells expressing sh4E-BP1 and/or eIF2α (S52A) were treated with IPZ (16 μM) for 24 h and subjected to western blot. h U87 cells from e were subjected to western blot. i U87 cells expressing sh4E-BP1 were treated as in a and subjected to western blot and flow cytometry. Results represent mean ± SD from three independent experiments. j U87 cells expressing shKPNB1s and/or sh4E-BP1 were treated as in b and subjected to western blot or flow cytometry. Results represent mean ± SD from three independent experiments. k U87 cells expressing shKPNB1s and/or shATF4 were subjected to real-time PCR and western blot. Results represent mean ± SD from three independent experiments. l U87 cells expressing shATF4 were treated with IPZ (16 μM) for 24 h and subjected to real-time PCR and western blot. Results represent mean ± SD from three independent experiments. GAPDH was used as the loading control. *P < 0.05