Figure 2. Bortezomib enhances UPR signaling under normoxia and hypoxia.
(A) ATF4 levels in HeLa cells exposed to normoxic or hypoxic conditions for 2h before treatment with DMSO or bortezomib for 6h. Thapsigargin (TH; 1μM, 4h) was used as a positivecontrol. CHOP levels in HeLa cells exposed to hypoxia 6h before treatment with DMSO or bortezomib for 12h. Thapsigargin (TH; 500nM, 12h) was used as a positive control. Nuclear protein was isolated and immunoblotted for ATF4 and CHOP; lamin A/C (loading control). (B) Active XBP-1 protein (XBP-1376aa) was induced in HeLa cells exposed to normoxia or hypoxia for 6h before treatment with DMSO or bortezomib for 18h. Thapsigargin (1μM, 12h) was used as a control. Nuclear protein was isolated and immunoblotted for XBP-1; lamin A/C (loading control). (C) HT1080-ATF4-Luc cells were treated with DMSO or bortezomib and luminescence was measured 8h later. (D) Left Panel: mice with HT1080-CMV-Luc tumors on the left flank and HT1080-ATF4-Luc tumors on the right flank were given IP injections of the vehicle or bortezomib (1mg/kg) and imaged for optical bioluminescence 8h later. Right panel: Quantitation of in vivo bioluminescence of mice shown on the left panel. Results are normalized to CMV-luc activity in untreated (vehicle-injected) animals.