Figure 2.

Suppression of ER stress-induced autophagy by 3-MA inhibits the UPR. (a) HEK293 cells were either non-treated (NT) or pretreated with 3-methyladenine (3-MA, 10 mM) or wortmannin (WM, 100 nM) for 1 h. The cells were then either subjected to NS for 2 h, TG (2 μM) or Tun (3 μM) treatment for 4 h, either in the absence or continuous presence of 3-MA or WM as indicated. Cell lysates were analyzed for levels of LC3-I conversion to LC3-II, CHOP, GRP78 and β-actin by Western blot using antibodies sequentially. (b) HEK293 or HeLa cells were treated the same as in (a). Cell lysates were analyzed for CHOP, ATF4, GRP78 and β-actin expression by Western blot using antibodies sequentially. (c) Same as (b) except the cells were pretreated with wortmannin. (d) HEK293 cells were non-treated (0 h) or pretreated with 3-MA (10 mM) for 1 h, then treated with Tun (3 μM), either in the absence or continuous presence of 3-MA for the indicated time. Cell lysates were analyzed for levels of phospho-JNK1 (p-JNK1), JNK1, phospho-eIF2α (p-eIF2α) and eIF2α Further, total RNA were extracted and analyzed for expression of Xbp-1, spliced Xbp-1 (Xbp-1s) and β-actin (as a control) by RT-PCR. (e) HEK293 cells were non-treated (0 h) or pre-treated with wortmannin (100 nM) prior to Tun treatment and assayed for Xbp-1 splicing as described in (d). For all panels, the experiments were repeated 2 to 3 times.