Extended Data Fig. 3: Proteomics analyses after IFNγ treatment of MD55A3 melanoma cells.

a, Volcano plot depicting overall changes in the proteome upon IFNγ treatment as observed by analysis of quantitative mass-spectrometry data. x axis: log-transformed fold change between IFNγ versus control conditions in MD55A3 cells; y axis: corresponding log-transformed adjusted P value calculated from three independent biological replicates. Highlighted in blue are proteins that are significantly differentially expressed. Both IFNγ-mediated induction of IDO1 and WARS and the immunoproteasome components are indicated. b, Left, box plots depicting log-transformed fold change in the levels of protein (red; average of three replicates) and mRNA (brown; average of two replicates) in IFNγ-versus control-treated cells. Proteins were grouped according to the number of asparagine (N), tyrosine (Y) or phenylalanine (F) residues in the protein sequence. Boxes depict first, second and third quartiles; whiskers depict the range excluding the outliers. Test: Wilcoxon test (two-tailed); NS, not significant, *P < 0.05. For asparagine box plots the actual P values are 0.7, 0.8, 0.055 for protein quantification (left) and 0.8,0.2, 0.013 for RNA quantifications (right) in the order shown. Middle, same as left but for number of tyrosine (Y) residues. Actual P values are 0.7, 0.86 and 0.02 for protein quantification (left) and 0.98, 0.13 and 0.14 for RNA quantifications (right) in the order shown. Right, same as middle but for number of phenylalanine (F) residues. Actual P values are 0.7, 0.97 and 0.23 for protein quantification (left) and 0.3, 0.015 and 0.0038 for RNA quantifications (right) in the order shown. c, Western blot analysis of ubiquitinylated proteins in total cell lysates of MD55A3 cells mock- or IFNγ-treated, which were additionally incubated minus or plus MG132 (n = 1). d, A panel showing proteins with increased abundance in total cell lysates of MD55A3 cells treated with MG132 versus controls. Log₂-transformed fold changes were calculated on data of three independent replicates. e, Box plots depicting log fold change in protein levels (average of three replicates) in IFNγ-treated versus control conditions in MD55A3 cells treated with proteasome inhibitor, for proteins stratified for different numbers of aspargine (N), tyrosine (Y) and phenylalanine (F) residues in their sequence. Boxes depict first, second and third quartiles; whiskers depict the range excluding the outliers. Test: Wilcoxon test (two-tailed); NS, not significant and *P < 0.05. Actual P values are 0.34, 0.44, 0.77 and 0.96, 0.84, 0.024 and 0.29, 0.84, 0.34 in the order shown. f, Box plots depicting protein length (number of amino acids) for stratified group of genes with increasing number of tryptophan (left) and asparagine (middle), with their ratios (right). g, Density of RPFs (average of two replicates) 300 codons across individual tryptophan codons (black line), two tryptophans that are present within a distance of 8 codons (green line) and two tryptophans that are present at a distance greater than 8 codons (red line). h, Box plot depicting bump scores (average of two replicates) for instances of two tryptophans separated by fewer than 8 codons (green) and more than 8 codons (red). Bump scores are calculated in MD55A3 cells. Boxes depict first, second and third quartiles; whiskers depict the range excluding the outliers. Test: Wilcoxon Ttest (two-tailed); ***P < 0.0005. Actual P value is < 2.2 ×10⁻¹⁶. i, Box plot depicting protein level changes (log-transformed fold change, average of three replicates) between IFNγ and control conditions. The graph represents genes that have two tryptophan codons within a distance of 8 codons (green) or genes having a distance of more than 8 codons between two tryptophans (red). Test: Wilcoxon test (two-tailed); ***P < 0.0005. Actual P value is < 2 ×10⁻¹⁶ j, Same as i, but for asparagine (N), in MD55A3 cells. Test: Wilcoxon test (two-tailed); NS, not significant. Actual P value is 0.83.