<p>(A) Huh7 cells were exposed to 100 nM bufalin for 12, 24, or 48 h. Untreated cells served as controls. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. (B) Huh7 cells were transfected with control, eIF2α, CHOP, or IRE1 siRNA for 24 h and then incubated with 100 nM bufalin for 24 h. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. The relative band density from control-siRNA transfected cells was defined as 1. (C) Huh7 cells were transfected with control or Akt siRNA for 24 h and then incubated with 100 nM bufalin for 24 h. Cell lysates were immunoblotted, and the density of each band was measured. Band densities were normalized to β-actin. The relative band density from control-siRNA transfected cells was defined as 1. (D) Huh7 cells were incubated with 100 nM bufalin and/or 5 μM sorafenib for 48 h. The cells were immunostained with Abs against IRE1 (red) and p-Akt (green) as well as with DAPI (cellular nuclei, blue). The data represent three independent experiments. N.S., not significant. “*” (P<0.05) and “**” (P<0.001) vs. untreated control; “##” represents P<0.001.</p
