Knockdown of ATF4 increases sensitivity to bortezomib. (A) Western blot analysis of ATF4 and Mcl-1 expression levels in bortezomib-treated U266 cells (2.5nM for 16 hours) and MMS1 cells (5nM for 16 hours). Top panels show the representative blots of 3 independent experiments; bottom panels show densitometric analysis. (B-C) qRT-PCR measurement of ATF4 downstream gene-expression levels after knockdown ATF4 in the presence of bortezomib in U266 cells (2.5nM for 16 hours; B) and MMS1 cells (5nM for 16 hours). n = 3 for all experiments. *P < .05 and **P < .01 versus si-mock samples. Data represent the means ± SD. (D) Knockdown of ATF4 induces apoptosis in U266 cells. ATF4 knockdown or control U266 cells were treated with 2.5nM bortezomib for 16 hours. Bortezomib was added in the medium 4 hours after electroporation-mediated RNAi. Apoptosis of each sample was examined by flow cytometry after annexin V–FITC/7-amino-actinomycin D staining. FACS data shown are representative of 3 independent experiments.