Extended Data Fig. 8: The cellular immunopeptidome is altered by PSME4 modulation.
a, The number of peptides identified in A549 cells 6 hours after acid stripping and stimulation with TNFα and IFNγ. Peptide count is shown both for cells depleted of PSME4 (KD) or control (Ctrl). Peptides were derived from n = 3 independent samples per treatment (Wilcoxon rank sum test; bars indicate mean ± s.d.). b, Principal component analysis (PCA) of the peptides identified and their abundances in the immunopeptidomics experiment (n = 3 independent samples). c, The mean fold change ratio between PSME4 depleted (KD) and control (WT) peptides is plotted for all the peptides identified. (One-sided T test of FC versus mu = 0) d, Whole cell proteomics was performed and a volcano plot for the change in protein abundance between the depleted (KD) and control (WT) conditions is shown (two-sided Student’s T test). e, Peptides are divided based on predicted haplotype and the fold change between PSME4 depletion (KD) is shown for each group (n = 3 independent samples). Box plots span the first to third quartiles and whiskers show 1.5× interquartile range. f, The fold change between KD and WT for the 179 peptides which end in K (orange line) was compared to 179 peptides randomly selected from the immunopeptidome 10,000 times (grey). g, A549 cells transfected with a PSME4-expressing plasmid or empty vector as a control. Cell lysates were blotted for PSME4. β-Actin was blotted as a loading control. Experiment was repeated twice with similar results h, Quantification of PSME4 band intensity in A549 cell line with PSME4 OE or Ctrl across n = 3 independent samples normalized to actin as a loading control (two-sided paired student’s t-test **P = 0.0045). i, Principal component analysis (PCA) of the peptides identified and their abundances in the immunopeptidomics experiment. j, Following ratio normalization to control for batch effect, the correlation between the ratios for the 463 altered peptides is shown across the replicates. k, The mean fold change ratio between overexpression (OE) and control peptides is plotted for the 463 peptides differentially presented upon PSME4 overexpression. l, The percentage of peptides with each indicated carboxy terminus is plotted for the peptides significantly increased in the PSME4 overexpression or empty vector control (denoted WT). m, The ratio between the enrichment score for the cellular component groups enriched in peptides increased in expression upon PSME4 overexpression (OE) over control (WT).
