Fig. 4: Immunopeptidomic analysis of HCT116 cells.
From: Long non-coding RNA-derived peptides are immunogenic and drive a potent anti-tumour response
A Immunopeptidomics analysis in T1-44 (1 µM for 48 h) or DMSO-treated HCT116 cells. The experiment was performed in biological duplicate. Indicated is the overlap of MHC-bound lncRNA-derived peptides identified from the qualitative analysis. 55 peptides were identified. B Peptide length of each human lncRNA-derived peptide is displayed as pie chart. 76 quantifiable peptides in total (pooled from two independent experiments) were detected (data derived from an immunopeptidomics experiment performed in biological independent replicate, each with 2 technical replicates. C (a) Sequence logos of amino acid conservation in lncRNA-derived peptides for each MHC allele; b) Predicted MHC allele frequency for identified lncRNA-derived peptides is displayed as a percentage of total. D Heatmap of lncRNA-derived peptide abundance from the quantitative immunopeptidomics analysis (76 peptides) (both up- and down-regulated in T1-44 treatment vs. DMSO). Relative abundance values were converted by normalisation to the mean. Yellow, up-regulation; blue, down-regulation; ivory, no change in abundance. n = 2 independent experiments (each with two technical replicates); E The percentage of lncRNA genes giving rise to MHC class I-bound peptides that score as potential direct E2F1 target genes, or are associated with other potential E2F1 target genes. Analysis was performed on all unique peptide-coding lncRNA genes identified. F (a) Example polysome profiling assay from HCT116 cells, indicating total RNA quantity detected in each collected fraction. (b) Polysome profiling assays for MALAT1 and AC079135.1 lncRNAs are displayed. Data are presented as percentage of total RNA in each fraction; n = 3 independent experiments (each with three technical replicates); G (a) Diagram of lncRNA ORF cloning strategy. The predicted ORF and potential endogenous ribosome binding site were inserted in frame with a 3xFLAG tag. A ribosome binding site is not provided in the vector itself. (b) Part of the MALAT1 and AC079135.1 lncRNA transcripts are displayed, with the predicted ORF (shown in red) giving rise to the identified MHC class I-bound peptide (boxed in black). Potential start residues are highlighted in red text. (c) Immune-fluorescence of HCT116 cells transfected with MALAT1 and AC079135.1 ORF-Flag plasmids. n = 2 independent experiments (d) Transfected HCT116 cells analysed by immunoblot. n = 3 independent experiments.
