Figure 4 : Nutlin 3 and 5-FU treatment increases MHC class I expression in a p53-dependent manner.

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

Figure 4

(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 β-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.