Figure 5 : Restoration of ERAP1 expression rescues the MHC class I expression.

From: p53 increases MHC class I expression by upregulating the endoplasmic reticulum aminopeptidase ERAP1

Figure 5

(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 β-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).