3.

Hypoxia, but not doxorubicin, initiates the ER stress response in H9c2 cells. (A) mRNA expression levels of the unfolded protein response (UPR) mediators XBP-1, GADD34 and ATF4 assessed by RT-PCR of total RNA samples isolated from cells exposed to hypoxia or 0.5 μM doxorubicin for the times indicated. (B) Alterations in the expression of ER stress associated proteins analysed by Western blotting using protein harvested after exposure to hypoxia or 0.5 μM doxorubicin for up to 48 hrs. Whole cell lysates were analysed for levels of Grp78 (10 μg) and CHOP (30 μg) and processing of pro-caspase-12 (30 μg). ER stress inducer thapsigargin (2 μM; 4 hrs for mRNA, 36 hrs for protein) was used as a positive control (+ve). displayed are the means ± S.D. of three independent experiments, *P < 0.05.