Figure 2. The amino acid sensor GCN2 is activated by uncharged tRNAs and is required for optimal activation of ATF4 upon MYC induction.

a. Representative immunoblot showing activation of GCN2 following MYC induction using cytoplasmic lysates. b. DLD-1: MycER cells were transfected with non-targeting siRNA or siRNA against PERK, GCN2 or both. ISR signaling and apoptosis were assessed by immunoblotting after MYC activation. c. Representative immunoblot analysis of cytoplasmic and nuclear lysates from GCN2 +/+, GCN2−/− and ATF4−/− MycER MEFs after 16hrs of MYC activation. d. DLD-1: MycER cells were pretreated with DMSO or 50μM RNA POLIII inhibitor (ML60218) for 2hrs prior to MYC activation. Indicated protein levels were measured by immunoblotting from cytoplasmic lysates. e. Microarray of aminoacyl–tRNAs of DLD-1: MycER cells after MYC induction at indicated times, four independent experiments, one-way ANOVA, *, p<0.05; **, p<0.01; ***, p< 0.001 (Refer to Statistics Source Data table for exact p values). (−) Leu denotes Leu-deprived cells and the marked tRNAs reading Leu codons are uncharged, n=1. tRNA probes are depicted with their cognate codon and the corresponding amino acid; Meti, initiator tRNA^Met. Two different probes recognizing two different tRNA^Leu isodecoders that pair to the same codon TTA/G Leu codon but differ in their sequence outside the anticodon were used on the arrays. Brackets show decreased charging of tRNA^Leu isodecoders in Leu deprivation condition. All immunoblots are representative of three biological replicates showing similar results. Unprocessed scans of blots are shown in Supplementary Fig 7.