Figure 4.

GSK2606414 Increases UPR Signaling through eIF2a-ATF4 while Decreasing Signaling through IRE1alpha and ATF6

(A) Illustration of tripartite UPR reporter design for live-cell imaging of IRE1a splicing of XBP1 intron sequence, ATF4 translation downstream of eIF2a, and activity of the ATF6 consensus DNA binding sequence. (B) 3D tumor spheroids containing reporter constructs were treated with 100 nM thapsigargin and 5 μM GSK2606414, and imaged at 72 h to profile lentiviral UPR reporter constructs. To aid clarity, grayscale images are presented using the pseudo-color scale shown. (C) 3D tumor spheroids containing reporter constructs were treated with reovirus and GSK2606414, and imaged at 72 h. (D) Automated image analysis was used to identify spheroids and calculate average reporter intensity. Each data point represents an independent experiment, each containing the average of at least four spheroids. (E) 2D cell lysates were collected after 48-h treatment of cells with reovirus and GSK2606414. Lysates were probed for pSer51 EIF2a. (F) Densitometry quantification of pSer51 eIF2a from western blots of three independent experiments. Data are corrected for loading and normalized to control. Data are ±SEM of a minimum of three independent experiments with statistical analysis by unpaired t test, *p < 0.05.