Fig 1: ERAP1 trims HBcAg-specific peptides in HepG2.2.15 cells. HepG2.2.15 cells were treated with ERPA1-IN-1 (50 µM) for 72 h. Cells were lysed, and the extracted protein was then analyzed by LC-MS/MS. The LFQ intensity of 8–16-mers residues was calculated and the peptides origin was identified by the MaxQuant (1.5.3.17) software via sequence-specific annotations based on the UniProt databases. ns, no significance; ***p < 0.001.
Fig 2: Nutlin 3 and 5-FU treatment increases MHC class I expression in a p53-dependent manner.(a) Real-time qPCR analysis of ERAP1 mRNA expression in Nutlin 3 (25 µM) or DMSO control-treated HCT116 (p53+/+) and HCT116 (p53-/-) cells. **P<0.01, NS, not significant (two-tailed Student’s t-test). (b) Western blot of ERAP1, p53, p21 and ß-actin (loading control) expression in Nutlin 3 or DMSO control-treated HCT116 (p53+/+) and HCT116 (p53-/-) cells. (c) Flow cytometric analysis of MHC class I (W6/32 labelling) expression in Nutlin 3 or DMSO control-treated HCT116 (p53+/+) (Red) and HCT116 (p53-/-) (Blue) cells after 24 and 48 h treatment. Cells incubated with fluorescence-labelled secondary antibodies alone served as background controls (Grey). (d) Immunofluorescence images of MHC class I expression, shown by W6/32 labelling, of Nutlin 3 or DMSO control-treated HCT116 (p53+/+) and HCT116 (p53-/-) cells at 48 h post treatment. DAPI and Brightfield images confirm the relatively equal number of cells captured. Scale bar, 50 µm. (e) Real-time qPCR analysis of ERAP1 mRNA expression in 5-FU (50 µg ml-1) or DMSO control-treated HCT116 (p53+/+) and HCT116 (p53-/-) cells. *P<0.05, NS, not significant (two-tailed Student’s t-test). (f) Flow cytometric analysis of MHC class I (W6/32 labelling) expression in 5-FU or DMSO control-treated HCT116 (p53+/+) (red) and HCT116 (p53-/-) (blue) cells at 48 h post treatment. Data are representative of three independent experiments and error bars represent s.d. of technical replicates (mean±s.d.), n=3, in a,e.
Fig 3: Restoration of ERAP1 expression rescues the MHC class I expression.(a) Effect of p53-specific siRNAs in decreasing p53 and ERAP1 mRNA in HCT116 (p53+/+) cells as measured by real-time qPCR. Data are representative of three independent experiments (mean±s.d.). (b) Western blot analysis of ERAP1, p53, p21 and ß-actin (loading control) expression in control siRNA (si-Ctrl) or p53 siRNAs (si-p53-1, si-p53-2) transfected HCT116 (p53+/+) cells at 48 h post transfection. (c) Immunofluorescence images of MHC class I expression shown by W6/32 staining in control siRNA (si-Ctrl) or pooled p53 siRNAs (si-p53) transfected HCT116 (p53+/+) cells at 48 h post transfection. DAPI and Brightfield images confirm the relatively equal number of cells captured. Scale bar, 50 µm. (d) Flow cytometric analysis of MHC class I (W6/32 staining) expression in control siRNA (si-Ctrl) or p53 siRNA (si-p53-1, si-p53-2) treated HCT116 (p53+/+) (red) cells at 48 h post treatment. Cells incubated with fluorescence-labelled secondary antibodies alone served as background controls (Grey). Dotted line marked the MFI (mean fluorescence intensity, calculated by FlowJo) of W6/32 signal observed for si-Ctrl-treated cells. (e) Flow cytometric analysis of MHC class I (W6/32 staining) expression when pooled p53 siRNAs (si-p53) were co-transfected with expression plasmids expressing ERAP1a or ERAP1b isoforms into HCT116 (p53+/+) cells. pcDNA3.1 control plasmid was used to balance the total amount of transfected DNA. (f) Expression of overexpressed ERAP1a or ERAP1b proteins in HCT116 (p53-/-) cells as shown by western blot. (g) Flow cytometric analysis of MHC class I (W6/32 staining) expression in ERAP1a or ERAP1b overexpressed HCT116 (p53-/-) (blue) cells. Dotted line marked the MFI of W6/32 signal observed for pcDNA3.1 plasmid control-transfected HCT116 (p53-/-) cells. The pcDNA3.1 control plasmid transfected HCT116 (p53+/+) (red) cells were also analysed for MHC class I expression. The calculated MFI values for all different treatments are shown in (h).
Fig 4: Influenza A virus infection enhances MHC class I expression via p53-activated ERAP1 in A549 (p53+/+) cells.(a) Real-time qPCR analysis of ERAP1 mRNA expression in mock control and H1N1 PR8-infected cells. Data are representative of three independent experiments (mean±s.d.). (b) Western blot of ERAP1 and ß-actin (loading control) protein expression at different time points in mock control and H1N1 PR8-infected cells. (c) Immunofluorescence microscopic images of p53 and ERAP1 expression in mock control and H1N1 PR8-infected cells at 48 hpi. Overlay images show co-localization/co-expression of p53 and ERAP1. (d) Immunofluorescence microscopic images of ERAP1 and MHC class I (W6/32) expression in mock control and H1N1 PR8-infected cells at 48 hpi. Overlay images showed co-localization/co-expression of ERAP1 and MHC class I. DAPI and Brightfield images confirm the relatively equal number of cells captured. Scale bar, 50 µm. (e) Flow cytometric analysis of MHC class I (W6/32) expression in p53 siRNA transfected or p53 siRNA and ERAP1 expression plasmid (ERAP1a or ERAP1b, 3 µg per well) co-transfected A549 (p53+/+) cells. Twenty four hours after transfection, cells were infected with H1N1 PR8 (MOI 0.5) or mock control and incubated for another 24 h. Dotted lines marked the MFI of W6/32 signal observed for control siRNA (si-Ctrl) transfected A549 (p53+/+) cells in the presence (PR8) or absence (mock control) of virus infection. The calculated MFI values for all different treatments are shown in (f).
Fig 5: ERAP1 is transcriptionally regulated by p53 via a bona fide p53RE.(a) Real-time qPCR validation of fold change in ERAP1 mRNA expression in HCT116 (p53-/-) cells transfected with wild-type p53 (p53WT) or one of 6 p53 mutants, compared with cells transfected with the pcDNA3.1 control. (b) Real-time qPCR analysis of relative basal ERAP1 mRNA expression levels in p53+/+ and p53-/- HCT116 cells. **P<0.01 (two-tailed Student’s t-test). (c) Western blot of ERAP1, ERAP2, p21, p53 and ß-actin (loading control) in HCT116 (p53+/+) and HCT116 (p53-/-) cells. (d) Dual-luciferase assay results of p53WT or the p53 dominant-negative mutant R175H co-transfected with different ERAP1 RE constructs (pGL3-pro-ERAP1-RE1 and RE2, sequences listed in the table at the bottom panel) into HCT116 (p53-/-) cells. Data are presented as percentage luciferase activity relative to the pcDNA3.1 control vector-transfected cells. Luciferase assay using p21 promoter construct (pGL3-pro-p21) serves as the positive control. (e) Binding affinity of wild-type p53 (p53WT) or the six p53 mutants to the identified ERAP1 p53RE sequences (wild-type or mutant) was determined by ProLabel Protein–DNA binding assay. (f) Schematic representation of identified ERAP1 p53RE (RE2) in relation to the ChIP-seq peaks and genomic localization of ERAP1 gene. TSS: transcription start site. Data are representative of three independent experiments and error bars represent s.d. of technical replicates (mean±s.d.), n=3, in a,b,d,e.
Supplier Page from Abcam for Anti-ARTS1/ERAP1 antibody [EPR6069]