Extended Data Fig. 1. Reporter assays identify IFNγ-induced W>F codon reassignment.

(a) A scheme of the reporter V5-ATF4^(1-63)-tGFP constructs used in this study. The tGFP gene was placed either in-frame or +1 nucleotide (nt) out-of-frame after the tryptophan codon at position 93 (W93). # and $ mark tGFP-containing and truncated protein products, respectively. (b) Dot plot depicting relative tryptophan levels of MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells treated with IFNγ (250U/mL) for 48 h. (c) MD55A3 melanoma cells expressing V5-ATF4^(1-63)-tGFP and V5-ATF4^(1-63)-tGFP⁺¹ were subjected to IFNγ treatment. Whole cell extracts were subjected to immunoblotting analyses using anti-V5, anti-tGFP, anti-Tubulin and anti-IDO1 antibodies. M: marker lane. Red arrows mark in-frame (#) and out-of-frame ($) products, as depicted in panel a. (n = 2 independent experiments). (d) Amino acid levels for tryptophan (W), tyrosine (Y), and phenylalanine (F) of the MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells depleted of the indicated amino acids, as in Fig. 1d. Each bar represents the average of 3 independent experiments +/- stdev. (e) MD55A3 cells expressing V5-ATF4^(1-63)-tGFP and V5-ATF4(1-63)-tGFP⁺¹ were depleted for tryptophan for 48 h and then treated or not with the proteasome inhibitor MG132 (10μM) as indicated in the scheme in panel b. Whole cell extracts were subjected to immunoblotting analyses using anti-V5, anti-tGFP, and anti-Tubulin antibodies. Red arrows mark in-frame (#) and out-of-frame ($) products. (n = 2 independent experiments). (f) Line plots depicting the peptides detected following V5-IP/MS of lysates of MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells from mock or IFNγ-treated conditions. The dashed line represents the site of the tryptophan 93 codon (W93) and the x-axis represents the in-frame part of the reporter protein before and after the tryptophan codon. (g) Dot-plot depicting log2 fold changes between mock and IFNγ-treated MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells for the tryptic peptide intensities presented. Each dot represents the intensity of a unique peptide. Lines represent the average intensities of all peptides +/-stdev. (h) The same analysis as performed in panel f, but for mock and tryptophan-depleted MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells. (i) The same analysis as performed in panel g, but for mock and tryptophan-depleted MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells. (j) Heatmap depicting a log2 fold change between mock and tryptophan-depleted MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells for amino acid substitution events for each of the amino acids in the tryptic peptide spanning the W93 codon. (k) Dot-plots depicting peptide intensity values in either mock (Ctrl) or amino acid depleted MD55A3-V5-ATF4^(1-63)-tGFP⁺¹ cells for the specific codon reassignments detected in the various depleted conditions, as indicated. Each dot represents an independent biological replicate and the line represents the average of the 3 replicates +/- standard deviation (stdev). (l-o) tGFP median intensity of reporter vectors encoding for tGFP, tGFP^F26W, or tGFP^F26A stably introduced into the indicated cell lines and conditions. Marked in dotted line is the highest value of the highest point in the background. Each dot represents a biological replicate and the line represents the average of the triplicates +/- stdev.