erap1 ic3  (MedChemExpress)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Analysis of the immunopeptidome of WT, inhibitor-treated, and ERAP1 KO A375 cells. Panel A , heat map from LC-MS/MS run 1 showcasing peptide distribution across different experimental conditions. Hierarchical clustering was calculated by Manhattan distance. Colors indicate peptide intensities, ranging from low ( red ) to high ( blue ). (Graph generated with Spectronaut v. 19). Panel B , principal component analysis (PCA) from LC-MS/MS run 1. Principal components 1 and 2 contribute to the explanation of 50% of the sample variability. Based on these two PCs, WT samples ( circle ), inhibitor-treated samples ( diamond ), and KO samples ( square ) formed three distinct groups, indicating that the replicates within each group share similarities with each other but there were differences in the immunopeptidomes between different treatments. Panels C – E , volcano plots from LC-MS/MS run 1 indicating the statistical significance of the differences between ( C ) the inhibitor-treated and the WT A375 cells, ( D ) the genetically modified (KO) and the WT A375 cells, and ( E ) the KO versus inhibitor-treated cells. Each circle represents a unique peptide sequence. Peptides with a q-value ≤0.05 and a log2 fold change ≥1 are considered statistically significant. ERAP, endoplasmic reticulum aminopeptidase.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Liquid Chromatography with Mass Spectroscopy, Generated, Genetically Modified, Sequencing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Binding affinity predictions and length distribution of binders. Panel A , distribution of predicted affinities (NetMHCpan-4.1) of the significantly upregulated or downregulated peptides after inhibitor treatment or ERAP1 KO for the HLA alleles present in A375 cells (A∗01:01, A∗02:01, B∗44:03, B∗57:01, C∗06:02, and C∗16:01). Each point on the graph signifies a distinct peptide sequence. Only the score for the top predicted HLA allele is depicted for each peptide. A set of random peptide sequences generated by RandSeq ( https://web.expasy.org/randseq/ ) was also plotted as negative control. Prediction scores below 2 ( dashed line ) indicate binding to at least one of the HLA alleles, with scores below 0.5 ( dotted line ) indicating strong binding. Panel B , length distribution of the significantly upregulated or downregulated binding peptides after inhibitor treatment or ERAP1 KO. ERAP, endoplasmic reticulum aminopeptidase; HLA, human leukocyte antigen.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Binding Assay, Sequencing, Generated, Negative Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Immunogenicity analysis of A375 cells. Panel A , representative fluorescence microscopy images of activated PBMCs ( red ) and caspase positive cells ( green ) in all conditions. Panel B , graph showing the changes in caspase-positive inhibitor-treated ( diamond ) and ERAP1 KO ( square ) A375 cells, compared to A375 WT cells ( circle ). The average, with its respective SD, was calculated from six images per experimental condition. Statistical significance was evaluated by one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism v.8. Panel C , graph showing the change in mean-specific lysis in inhibitor-treated and ERAP1 KO cells in the PBMC cytotoxicity assay. The average, with its respective SD, was calculated from three technical replicates per experimental condition. The calculated adjusted p values comparing KO cells and inhibitor-treated cells to the WT cells are indicated. Statistical significance was evaluated by one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism v.8. ERAP, endoplasmic reticulum aminopeptidase; PBMC, peripheral blood mononuclear cell.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Immunopeptidomics, Fluorescence, Microscopy, Lysis, Cytotoxicity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Proteomic analysis of WT, inhibitor-treated, and ERAP1 KO A375 cells. Panel A , heat map showing the cluster analysis and distribution of proteins in the three conditions (WT, inhibitor-treated, and ERAP1 KO A375 cells) for each replicate (four biological replicates, each measured in three technical replicates) (graph generated with Perseus v. 1.6.15). Panel B , principal component analysis (PCA) of the three experimental conditions. Each point represents a distinct biological or technical replicate. Panels C and D , Hawaii plots of identified proteins, indicating the statistical significance of the observed differences in protein abundance between the two treatment conditions (inhibitor-treated and ERAP1 KO) and the WT cells. Three hundred twenty-five proteins in the inhibitor-treated ( C ) and 1095 proteins in the KO cells ( D ) were differentially expressed. Select proteins that participate in antigen presentation are indicated in red (graph generated with Perseus v. 1.6.15). Panels E , F , and G , heat maps of proteins participating in protein folding ( E ), proteasomal protein catabolic process ( F ), and protein processing in the endoplasmic reticulum ( G ) (graphs generated with Perseus v. 1.6.15). ERAP, endoplasmic reticulum aminopeptidase.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Generated, Quantitative Proteomics, Immunopeptidomics

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Dichloro fluorescein assay for measurement of reactive oxygen species. Panel A , representative images of WT, inhibitor-treated (INH), and ERAP1 KO A375 cells incubated with H2DCFDA ( left ) or Hoechst 33342 ( right ). Panel B , total fluorescence intensity per number of nuclei quantitated by direct cell imaging on a Cytation-5 instrument for the three biological conditions. Panel C , relative DCF fluorescence units (RFU) normalized to Hoechst dye fluorescence measured by direct fluorescence measurement on a BioTek Synergy H1 plate reader. The calculated adjusted p values comparing KO cells and inhibitor-treated cells to the WT cells are indicated. Statistical significance was evaluated by one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism v.8. ERAP, endoplasmic reticulum aminopeptidase.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Incubation, Fluorescence, Imaging

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Thioflavin T assay for ER stress. Panel A , representative images of WT, inhibitor-treated (INH), and ERAP1 KO A375 cells incubated with thioflavin T with or without treatment with DTT, as described in the Experimental procedures section. Panel B , total fluorescence intensity normalized for cell area for the three biological conditions in the absence or presence of DTT. Statistical significance was evaluated by one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism v.8. ER, endoplasmic reticulum; ERAP, endoplasmic reticulum aminopeptidase.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: ThT Assay, Incubation, Fluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Mitotracker assay for measurement of mitochondrial membrane potential. Panel A , representative fluorescence microscopy images of A375 cells (WT, inhibitor-treated, or ERAP1 KO) visualized after staining with DAPI ( blue ) and Mitotracker ( red ) as well as merged. Panel B , total fluorescence intensity of Mitotracker per identified nuclei for the three conditions. The calculated adjusted p values comparing KO cells and inhibitor-treated cells to the WT cells are indicated. Statistical significance was evaluated by one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism v.8. ERAP, endoplasmic reticulum aminopeptidase.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Membrane, Fluorescence, Microscopy, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: ERAP1 Activity Modulates the Immunopeptidome but Also Affects the Proteome, Metabolism, and Stress Responses in Cancer Cells

doi: 10.1016/j.mcpro.2025.100964

Figure Lengend Snippet: Seahorse extracellular flux analysis of effects of ERAP1 disruption on cellular metabolism. Panel A , Oxygen Consumption Rate (OCR) followed as a function of time during the experiment for WT, inhibitor-treated and ERAP1 KO A375 cells. Arrows at specific time points indicate the addition of glucose, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A to probe different metabolic components, as indicated by the colored regions in the figure. Panels B – G , quantification and comparison of components of mitochondrial metabolism between the three experimental conditions. Panel H , extracellular Acidification Rate (ECAR) followed as a function of time as in panel A. Panels I – L , quantification and comparison of basal glycolytic levels, spare glycolytic capacity, maximum glycolytic capacity and non-glycolytic acidification as calculated form the ECAR measurements. Statistical significance was evaluated by one-way ANOVA, followed by Dunnett’s multiple comparisons test in GraphPad Prism v.8.

Article Snippet: THP-1 WT (TIB-202, American Type Culture Collection) and THP-1 ERAP1 KO cells, generated in Dr Doriana Fruci’s lab at Ospedale Pediatrico Bambino Gesù (Król et al , manuscript in preparation), were cultured in Rosswell Park Memorial Institute 1640 (Biowest, L0501) with the addition of 10% FBS (Biowest, S1810), 2 mM L-glutamine (Biowest, X0550), and 1% Penicillin-Streptomycin (Biowest, L022) as usual at 37 °C, 5% CO 2.

Techniques: Disruption, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Controlling intracellular processing to enhance spherical nucleic acid immune stimulation

doi: 10.1073/pnas.2409554122

Figure Lengend Snippet: Intracellular trafficking of the antigen on NC SNA and ERAP1-responsive SNAs over time by confocal microscopy on JAWS II. ( A ) Scheme of ERAP1-responsive SNAs. TLR9 agonist CpG DNA (purple) containing a cholesterol anchor on the 3’ end is duplexed with a complementary strand (orange) that is conjugated to a peptide (green) containing EPL and SIINFEKL via a non-cleavable N-β-maleimidopropyl-oxysuccinimide ester (BMPS) crosslinker (black). The intercalation of this duplex into the liposome forms the SNA. Manders’ colocalization coefficients of the fraction of ( B ) early endosome (EEA1), ( C ) lysosome (LAMP1), or ( D ) ERAP1 (ARTS1), which are labeled with antibodies, is colocalized with the antigen labeled with TAMRA on NC, LAMM, or LAKK SNA from 2 to 16 or 20 h. A two-way ANOVA with Tukey’s multiple comparisons test between all groups of SNA was performed. ( E ) Images of SIINFEKL peptide (TAMRA, red) colocalized with ERAP1 (Alexa-488, green) delivered by NC, LAMM, or LAKK SNA from 2 to 20 h. (Scale bar, 10 µm.) For panels displaying multiple cells, the cell of interest is indicated with an arrow. All analysis values are an average of 8 representative images. Data presented as mean ± SEM ( B – D ). **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05.

Article Snippet: The inhibitors used for this experiment are ERAP1-IN-1 (MedChemExpress HY-133125) for ERAP1, leupeptin (Thermo Fisher 78435) for endolysosomal proteases, and LHVS (Sigma Aldrich SML2857) for CatS.

Techniques: Confocal Microscopy, Labeling