Figure 4.

Selective activation of the ISR following BIRC6 depletion. A, Effects of BIRC6 depletion on gene expression. RNA samples were harvested 4 days after the transduction of either a control sgRNA (sgCh2-2) or an sgRNA targeting BIRC6 (sgBIRC6). The gene-level expression change [log-fold changes, or LogFC (sgBIRC6/sgCh2-2)] and the significance of the observed change [−log₁₀ (P)] were plotted separately for the three dependent models and the three nondependent models. Green dots represent significant changes (adjusted P value < 0.01). B, Gene set enrichment analysis for the differentially expressed genes. The positions of the circles indicate the enrichment score for the individual hallmark gene sets, while the sizes of the circles reflect the significance of enrichment. These analyses were performed in HCC202 breast cancer cells and SNU503 colon cancer cells. C, Activation of p-eIF2a/ATF4 signaling following BIRC6 depletion in the dependent cell lines. The Cas9-expressing derivatives of the indicated cells were transduced with the indicated sgRNA, and their lysates were harvested 4 and 7 days later. The cell lysates were treated with arsenite (300 μmol/L, 3 hours), thapsigargin (1 μmol/L, 6 hours), or a vehicle control (DMSO). These lysates were subjected to immunoblotting for markers of the ISR, including p-eIF2S1, ATF4, and ATF3. Values represent the intensity of the p-eIF2α band relative to that of corresponding t-eIF2α band. D, Differential expression of the target genes for three different signaling arms of the UPR response, PERK–p-eIF2a/ATF4 pathway, ATF6 pathway, and IRE1/XBP1 pathway. The LogFCs in the expression levels of the individual transcriptional targets of these three signaling arms, observed in the RNA sequencing experiment shown in A, are indicated. ns, P ≥ 0.05; ***, P < 0.001; ****, P < 0.0001 (dependent vs. nondependent; LogFCs of the target genes that are specific only to the PERK–p-eIF2a/ATF4, ATF6, or IRE1/XBP1 pathway were compared between these two groups of cell lines). E, Schematic of the ISR. The four members of the EIF2AK family of kinases (GCN2, PKR, HRI, and PERK) are activated by discrete types of stress stimuli. However, their activation converges on the phosphorylation of eIF2a, resulting in the global shutdown of protein synthesis and selective induction of a subset of proteins including ATF4. The RNA sequencing experiment (A, B, D) was conducted once, while the experiment shown in C was conducted twice.