Figure 7: Influenza A virus infection enhances MHC class I expression via p53-activated ERAP1 in A549 (p53+/+) cells.
From: p53 increases MHC class I expression by upregulating the endoplasmic reticulum aminopeptidase ERAP1
(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 β-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).
