Fig. 2. KPNB1 inhibition increases total and cell surface DR5 level in glioblastoma cells.

a–d Intact shATF4-expressing U87 cells either expressing shKPNB1s (a) and (b) or treated with IPZ (16 μM) for 24 h (c) and d were stained with APC anti-human DR5 antibody or IgG isotype ctrl antibody. Cell surface DR5 levels were measure by flow cytrometry a and c. Mean fluorescence intensity (MFI) was shown in b and d. Results represent mean ± SD from three independent experiments. *P < 0.05. e Western blots analysis of cytosolic and nuclear DR5 level in shKPNB1-expressing U87 cells. Equal amount of cytosolic and nuclear protein was loaded. f–h U87 and U251 cells were harvested after shKPNB1-encoding lentiviruses infection (f) or IPZ (16 μM) treatment (g) for indicated times or after treating with various concentration of IPZ for 24 h (h) Levels of proteins were analyzed by western blot. i, j Intact U87 and U251 cells either expressing shKPNB1s or treated with IPZ (16 μM) for 24 h were stained with APC anti-human DR4 antibody or IgG isotype ctrl antibody. Cell surface DR4 levels were measured by flow cytometry (i). MFI was shown in (j). Results represent mean ± SD from three independent experiments. *P < 0.05. N.S. not significant. k Western blot analysis of DR4 and DR5 in shKPNB1-expressing A172, U87, U118, U251 and HA cells. l Western blot analysis of DR4 and DR5 in U87, U118 and U251 cells treated with IPZ (16 μM), IVM (16 μM) or INI-43 (8 μM) for 24 h. GAPDH was used as the loading control of total cell lysates or cytosolic fraction. Lamin B was used as the loading control of nuclear fraction