FIG.7.

ER stress sensitizes cells to TNF-α-induced cell death. (A) MCF-7 cells were treated with or without thapsigargin or tunicamycin for 4 h. After being washed with medium (Dulbecco's modified Eagle medium + 10% FBS) two times, cells were incubated without or with TNF-α (30 ng/ml). Cell death was quantified by MTT assay. Data are representative of triplicate experiments. (B) ER stress inhibits TNF-α-induced activation of NF-κB. MCF-7, L929, and DU145 cells were treated with DMSO, thapsigargin, or tunicamycin for 4 h and then stimulated with TNF-α (15 ng/ml) for the indicated times. Cell lysates were prepared, and 20 μg of protein from each sample was used for Western blotting with anti-IκBα and anti-β-actin. (C) ER stress induces degradation of TRAF2. MCF-7 and L929 cells were treated with thapsigargin or tunicamycin. Cell lysates were prepared, and 20 μg of protein from each sample was used for Western blotting with anti-TNFR1, anti-TRADD, anti-RIP, anti-TRAF2, anti-IKKβ, and anti-IKKγ. (D) ER stress inhibits TNF-α-induced activation of JNK. The same samples from panel D were used for immunoblotting with anti-JNK and anti-phospho-JNK antibodies. (E) ER stress does not influence transcription of TRAF2. MCF-7 cells were treated with thapsigargin or tunicamycin for 2 and 4 h. Total RNA was collected, and the mRNA level of TRAF2 was measured by RT-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) ER stress decreases stability of TRAF2. L929 cells were incubated with CHX (20 μM) or CHX plus thapsigargin (2 μM) for the indicated time, and then the TRAF2 protein level was examined by Western blotting. (G) Knockdown of TRAF2 protein sensitizes cells to ER stress-induced cell death. After transfection with TRAF2 siRNA or control siRNA for 48 h, MCF-7 cells were subjected to thapsigargin or tunicamycin for the indicated times. Cell death was assessed by an MTT assay. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.