Figure 5
Delaying the attenuation of IRE1 RNAse activity affects cell fitness in the face of repeated, transient ER stress. (A) MCF10A cells were induced for ER stress with 500ng/ml tunicamycin for 4 h; washed out and allowed to recover either on vehicle-containing fresh medium (DMSO), PP242-containing medium (500 nM) or Torin1-containing medium (250 nM), and extracted for total mRNA for semiquantitative RT-PCR for the relative amounts of XBP1 mRNA species. (B) MCF10A cells were treated as indicated (PP242: 500 nM; Torin1: 250 nM), and whole cell lysates were analyzed for the levels of phosphorylated AKT or total AKT, as a readout of net TOR kinase activity. (C) A schematic depiction of the treatment regime is shown. MCF10A cells were exposed to 500ng/ml tunicamycin for 4 h to induce ER stress, then washed and allowed to recover for 8 h in fresh medium containing vehicle (DMSO) or mTOR kinase inhibitor (PP242; 500 nM), and further washed out and cultured for 12 h until the next treatment round. (D) 6 independent biological replicates were analyzed. Cells were trypsinized and resuspended in equal volumes, and counted using an automated live cell counter (Countess, Invitrogen).
