Fig. 4: The stability and degradation pathways of ncORF peptides with different PepScores.

a PepScores and peptide lengths of 29 selected uORFs. b The PhyloCSF scores of 29 selected uORFs. c, d The ectopic expression of uORFs in HEK293T cells. Cells with ORF-Flag expression were stained with anti-Flag (green) and DAPI (blue). Empty vector and GFP-Flag were used as the negative and positive controls, respectively. Representative images of cells expressing selected uORFs are shown in (c). Scale bar, 50 μm. The y-axis represents the normalized fraction of cells expressing ORF-Flag. Data are shown as mean values ± SD of five (nontreatment) or four (MG132 treatment) replicates and are representative of three independent experiments. e Comparing peptide expression of uORFs with high vs. low PepScores in untreated cells. The P-value calculated using the two-sided Wilcoxon Rank Sum Test is shown. f The ROC curve measuring the performance using PepScore to classify uORFs with expression >5% vs. others. The AUROC value is shown. g The AUROC values obtained when using PepScore, peptide length, and PhyloCSF to classify highly vs. lowly expressed uORF peptides. h Compounds used in this study and their targeted pathways. i Comparing peptide expression of uORFs with high vs. low PepScores in MG132-treated cells. The P-value calculated using the two-sided Wilcoxon Rank Sum Test is shown. j–k The uORF peptide expression levels were analyzed using untreated cells or those treated with proteasome inhibitors or/and lysosome inhibitors. Representative immunostaining images of selected uORF peptides in each condition are shown in (j). Scale bar, 50 μm. The expression levels are shown in (k). Error bars represent the standard deviation of four replicates. l PepScores and peptide lengths of five selected ouORFs, one iORF, and two dORFs. m The PhyloCSF scores of selected ncORFs. n The ectopic expression levels of the noncanonical peptides. The calculation method is the same as in (d). k, n Data are shown as mean values ± SD of four replicates and are representative of three independent experiments. The peptide expression level can be found in Supplementary Data 9.