Journal: Nature Communications

Article Title: Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

doi: 10.1038/s41467-020-16588-9

Figure Lengend Snippet: a Viability at 72 h after drug treatment; data are represented as a fraction (%) of the DMSO control. Calculated IC 50 s: SKMEL5 = 12.74 μM, SKMEL28 = 14.62 μM, SKMEL2 = 16.98 μM, and IPC298 = 10.62 μM. Data are presented as mean values ± SD for n = 3 experimental replicates for all cell lines, except SKMEL5 ( n = 4). b Experimental setup of TMT-labeled immunopeptidomics experiments in melanoma cell lines. c Flow cytometry measurements of surface HLA expression in SKMEL5 cells. Data are represented as % of maximum signal, and the distributions are representative of three independent experiments. d Histogram distribution of log 2 fold change (FC) of (palbociclib/DMSO) for unique pMHCs, where FC is calculated from the mean intensity of n = 3 biological replicates per condition. Data are represented as a % of total unique peptides identified. e Volcano plot displaying log 2 (FC) of 1 μM treated SKMEL5 cells versus significance (mean-adjusted p value, unpaired two-sided t test). Colored points ( p < 0.05, log 2 (FC) > 1.56) correspond to processes in f . f Log 2 (FC) of significantly enriched peptides from GO term enrichment processes labeled with source protein name. False discovery rate (FDR)-adj. p value < 0.05. g Four-way Venn diagram of the number of source proteins of peptides significantly enriched (top value) or significantly increasing (bottom value) with 1 μM palbociclib. h Log 2 (FC) of pIRS2 peptide following 1 μM (gray) or 10 μM (black) palbociclib, * p < 0.05, unpaired two-sided t test. i Source protein name, peptide sequence, and log 2 (FC) of TAAs in SKMEL5 (left) and IPC298 (right) cells. Exact significance values and other source data are reported in the Source Data File.

Article Snippet: SKMEL5, SKMEL28, and MDA-MB-231 cell lines were obtained from ATCC (ATCC HTB-70, ATCC HTB-72, and HTB-26, respectively) and maintained in DMEM (Dulbecco’s modified Eagle’s medium) (Corning).

Techniques: Control, Labeling, Immunopeptidomics, Flow Cytometry, Expressing, Sequencing

Journal: Nature Communications

Article Title: Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

doi: 10.1038/s41467-020-16588-9

Figure Lengend Snippet: a Normalized enrichment score (NES) of significantly enriched pathways with 10 μM palbociclib, where +/− NES scores reflect enrichment directionality. For all, q < 0.25, and * p < 0.05, ** p < 0.01, and *** p < 0.001. b , c String network of protein–protein interactions of all source proteins from E2F peptides ( b ) significantly decreasing with 10 μM palbociclib, and OxPhos peptides ( c ) significantly increasing with 1 μM palbociclib for all cell lines, except SKMEL28, where peptides from 10 μM are depicted. Node color corresponds to cell line. d Quantification ( n = 9) of MitoTraker green intensity normalized to cell number following 72 h palbociclib treatment. Data are represented as a box and whiskers plot, with whiskers displaying minimum and maximum signal. Significance was determined using Dunnett’s multiple comparisons test for each condition versus DMSO. * p < 0.05 and **** p < 0.0001. e Correlation between log 2 fold change (FC) of (palbociclib/DMSO) for RNA expression ( y -axis) and pMHC presentation ( x -axis) of SKMEL5 cells treated for 72 h with 1 μM palbociclib, r 2 = 0.04. FC is calculated from the mean intensity of n = 3 biological replicates per condition. f Significantly enriched pathways using RNA-seq data ( p < 0.05, q < 0.25). Annotated pathways reflect pathways also identified in the immunopeptidome analysis (blue), and those that match with previous reported data (red) . g Log 2 (FC) for SKMEL5 OxPhos peptides significantly increasing ( p < 0.05, blue) with 1 μM palbociclib, and matched log 2 (FC) of RNA expression (black). Significant differences in RNA expression (palbociclib versus DMSO) are indicated. ** p < 0.01 and **** p < 0.0001 (Wald test, Benjamini–Hochberg (BH) adjusted). Exact significance values and other Source data are reported in the Source Data File.

Article Snippet: SKMEL5, SKMEL28, and MDA-MB-231 cell lines were obtained from ATCC (ATCC HTB-70, ATCC HTB-72, and HTB-26, respectively) and maintained in DMEM (Dulbecco’s modified Eagle’s medium) (Corning).

Techniques: Protein-Protein interactions, RNA Expression, RNA Sequencing

Journal: Nature Communications

Article Title: Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

doi: 10.1038/s41467-020-16588-9

Figure Lengend Snippet: a RNA-seq (black) and pMHC (blue) log 2 fold change (FC) of 1 μM palbociclib/DMSO, calculated from the mean intensity of n = 3 biological replicates per condition, for antigen-processing genes. b Surface HLA expression via flow cytometry of cells treated with 72 h IFN-γ shown as log 2 (FC) (IFN-γ/DMSO). Errors bars represent ± SD, biological replicates are n = 8, 11, 9, and 9 for SKMEL5, SKMEL28, SKMEL2, and IPC298, respectively. c Immunopeptidome log 2 (FC), dotted lines display quartiles, and mean fold changes (solid line) are 2.42, 2.50, 2.08, and 3.04 for SKMEL5, SKMEL28, SKMEL2, and IPC298 cells, respectively. d Significantly enriched pathways in SKMEL5 cells with 72 h IFN-γ, q < 0.25, * p < 0.05, ** p < 0.01. e Enrichment plot of IFN-γ response enrichment in SKMEL5 cells displays running enrichment score (green, right y -axis), and the log 2 (FC) (left y -axis) versus rank ( x -axis) for each peptide (gray). Open circles show significantly enriched IFN-γ peptides. f Volcano plot of IFN-γ-induced changes in SKMEL5 cells. Peptides are presented as the log 2 (FC) versus mean-adjusted p value (unpaired two-sided t test). Red points represent peptides significantly enriched ( p < 0.05, fold change > 2.42). g Venn diagram of significantly enriched source proteins (black) or peptides (gray) between IFN-γ and 1 μM palbociclib-treated SKMEL5 cells. h Protein–protein interaction network of significantly enriched source proteins in common, annotated by enriched gene ontology cellular components (CCs) and/or biological professes (BPs). Significance values are false discovery rate (FDR) adjusted. Exact significance values and other Source data are reported in the Source Data File.

Article Snippet: SKMEL5, SKMEL28, and MDA-MB-231 cell lines were obtained from ATCC (ATCC HTB-70, ATCC HTB-72, and HTB-26, respectively) and maintained in DMEM (Dulbecco’s modified Eagle’s medium) (Corning).

Techniques: RNA Sequencing, Expressing, Flow Cytometry