Figure 1.
Selective and specific activation of PERK signaling.
(A) Parental wild-type and transgenic HEK293 cells expressing the AP20187-sensitized Fv2E-Perk allele were treated for the indicated times with AP20187 (2 nM). Fv2E-Perk, Gadd34, and Chop mRNA levels were measured by quantitative PCR, normalized to levels of a housekeeping gene, Rpl19, and are shown relative to levels in untreated cells. Fv2E-PERK, phospho-eIF2α, and ATF4 proteins were detected by immunoblotting. Total eIF2α protein was measured as a loading control. (B) Parental wild-type and transgenic HEK293 cells expressing the AP20187-sensitized Fv2E-Perk allele were treated for the indicated times with AP20187 (2 nM). Xbp1 mRNA splicing was assesed by RT-PCR. The unspliced (u) and spliced (s) Xbp1 mRNA products are indicated as labeled. The asterisk indicates the position of a hybrid amplicon.
Figure 2.
Selective and specific activation of IRE1 signaling.
(A) Parental wild-type and transgenic HEK293 cells expressing the Ire1[I642G], or Ire1[I642G/K599A] allele were treated for the indicated times with 1NM-PP1 (1 µM), and wild-type cells were treated for 4 hours with thapsigargin (tg) (300 nM). IRE1[I642G] and IRE1[I642G/K599A] protein was detected by immunoblotting for the FLAG epitope. GAPDH levels were assessed as a protein loading control. Xbp1 mRNA splicing was determined by RT-PCR. The unspliced (u) and spliced (s) Xbp1 mRNA products are indicated as labeled. ERdj4 mRNA levels were measured by quantitative PCR, normalized to Rpl19 mRNA levels, and are shown relative to levels in untreated cells. (B) Parental wild-type and transgenic HEK293 cells expressing the Ire1[I642G] or Ire1[I642G/K599A] alleles were treated for the indicated times with 1NM-PP1 (1 µM); wild-type cells were also treated for 4 hours with thapsigargin (tg) (300 nM). ATF4 protein was detected by immunoblotting. GAPDH levels were assessed as a protein loading control.
Figure 3.
Sustained Perk signaling impairs cell proliferation.
(A) Parental wild-type and isogenic HEK293 cells expressing Fv2E-Perk, Ire1[I642G], or Ire1[I642G/K599A] alleles were treated with tunicamycin (5 µg/ml), 1NM-PP1 (1 µM), or AP20187 (2 nM), videographed for 48 hours, and frames from indicated time points are shown. Magnification bar, 125 µm. (B) Parental wild-type and transgenic HEK293 cells expressing the Fv2E-Perk allele were treated with AP20187 (2 nM), counted, and are shown relative to numbers of mock-treated cells at the indicated times. (C) Parental wild-type and transgenic HEK293 cells expressing Ire1[I642G] or Ire1[I642G/K599A] alleles were treated with 1NM-PP1 (1 µM), counted, and are shown relative to numbers of mock-treated cells at the indicated times.
Figure 4.
Sustained Perk signaling promotes apoptosis.
Parental wild-type and isogenic HEK293 cells expressing Ire1[I642G] or Fv2E-Perk were treated with thapsigargin (300 nM); 1NM-PP1 (1 µM); or AP20187 (2 nM) for the indicated times. Cleaved PARP protein was assessed by immunoblot. GAPDH protein levels served as a loading control.