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

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1 Supplementary Information p53 increases MHC class I expression by upregulating the endoplasmic reticulum aminopeptidase ERAP1 Bei Wang 1, Dandan Niu 1, Liyun Lai 1, & Ee Chee Ren 1,2 1 Singapore Immunology Network, A*STAR, 8A Biomedical Grove, Singapore Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Correspondence and requests for materials should be addressed to E.C.R. ( ren_ee_chee@immunol.a-star.edu.sg) Supplementary Figures S1-S8 Supplementary Tables S1-S4 1

2 Supplementary Figures Supplementary Figure S1. Functional characterization of 6 p53 mutants used in this study. (a-f) Correlation analysis of relative fold change in luciferase activity for p53wt and each of the 6 p53 mutants relative to the pcdna3.1 control using background LASP1 gene promoter luciferase constructs containing 16 different dinucleotide core combinations. R: correlation coefficient. (g) Differential DNA binding activity of wildtype p53 and 6 p53 mutants to wild-type p53re sequence of p21 gene as determined by ProLabel Protein-DNA binding assay. Data are representative of three independent experiments (mean ± s.d.). 2

3 Supplementary Figure S2. Nutlin 3 treatment enhances MHC class I expression in a p53-dependent manner in osteosarcoma cell lines. (a) Real-time qpcr analysis for ERAP1 mrna expression in Nutlin 3 (25μM) or DMSO control treated U2OS (p53 +/+ ) and Saos-2 (p53 -/- ) cells. Data are representative of three independent experiments (mean ± s.d.). *p<0.05, n.s. not significant (two-tailed student s t-test). (b) Flow cytometric analysis of MHC class I (W6/32 labeling) expression on Nutlin 3 or DMSO control treated U2OS (p53 +/+ ) (Magenta) and Saos-2 (p53 -/- ) (Cyan) cells at 48h post treatment. Cells incubated with fluorescencelabeled secondary antibodies alone served as background controls (Grey). (c) Immunofluorescence images of MHC class I expression shown by W6/32 labeling of Nutlin 3 or DMSO control treated U2OS (p53 +/+ ) and Saos- 2 (p53 -/- ) cells at 48h post treatment. DAPI and Brightfield images confirm the relatively equal number of cells captured. Scale bar, 50μm. 3

4 Supplementary Figure S3. p53 specific sirnas down-regulate ERAP1 and diminish MHC class I expression in U2OS (p53 +/+ ) cells. (a) Effect of p53 specific sirnas (40nM) on decreasing p53 and ERAP1 mrna in U2OS (p53 +/+ ) cells, measured by real-time qpcr. Data are representative of three independent experiments and error bars represent standard deviation of technical replicates (mean ± s.d.), n=3. (b) Flow cytometric analysis of MHC class I expression (W6/32 labeling) in control sirna (si-ctrl) or p53 specific sirnas (si-p53-1, si-p53-2) transfected U2OS (p53 +/+ ) (Magenta) cells. Dotted line marks the MFI of the W6/32 signal observed for control sirna transfected U2OS (p53 +/+ ) cells. Cells incubated with fluorescence-labeled secondary antibodies alone served as background controls (Grey). (c) MFI quantified by FlowJo for flow cytometric analysis shown in (b). (d) Immunofluorescence images of MHC class I expression (W6/32 labeling) in control sirna (si-ctrl) or pooled p53 sirna (si-p53) transfected U2OS (p53 +/+ ) cells at 48h post transfection. DAPI and Brightfield images confirm the relatively equal number of cells captured. Scale bar: 50μm. 4

5 Supplementary Figure S4. Restoration of ERAP1 expression rescues MHC class I expression in Saos-2 (p53 -/- ) cells. (a) Western blot of ERAP1, p21, p53 and β-actin (loading control) in U2OS (p53 +/+ ) and Saos-2 (p53 -/- ) cells. (b) Overexpressed ERAP1a or ERAP1b proteins in Saos-2 (p53 -/- ) cells by western blot. (c) Flow cytometric analysis of MHC class I (W6/32 labeling) expression in ERAP1a or ERAP1b overexpressing Saos-2 (p53 -/- ) (Cyan) cells. Dotted line marks the MFI of the W6/32 signal observed for pcdna3.1 plasmid control transfected Saos-2 (p53 -/- ) cells. The pcdna3.1 control plasmid-transfected U2OS (p53 +/+ ) (Magenta) cells were also analyzed for MHC class I expression. Cells incubated with fluorescence-labeled secondary antibodies alone served as background controls (Grey). (d) The calculated MFI for flow cytometric analysis shown in (c). 5

6 Supplementary Figure S5. Overexpression of wild-type p53 up-regulates ERAP1 and enhances MHC class I expression in HCT116 (p53 -/- ) cells. (a) Time-course mrna expression analysis for the ERAP1 gene in p53wt or R175H mutant construct transfected HCT116 (p53 -/- ) cells. Data were presented as the fold expression relative to that in pcdna3.1 control plasmid-transfected cells. The measurement for p21 mrna level serves as positive control. (b) Flow cytometric analysis of MHC class I expression (W6/32 labeling) in p53wt or R175H overexpressed HCT116 (p53 -/- ) (Blue) cells. Dotted line marked the MFI of W6/32 signal observed for pcdna3.1 control plasmid transfected cells. (c) The calculated MFI for flow cytometric analysis shown in (b). 6

7 Supplementary Figure S6. H1N1 PR8 infection of A549 (p53 +/+ ) cells. (a) Cytopathic effect (CPE) of human A549 cells infected with H1N1 PR8. Human A549 cells treated with mock control (infecting medium only) exhibited a cobblestone appearance (top left panel). The CPE could be observed at Day 5 post infection in H1N1 PR8 infected cells with multiplicity of infection level of 0.5 (top right panel), 1.0 (bottom left panel) and 2.0 (bottom right panel). (b) The mrna expression of the virus NS1 (non-structural protein 1) gene in mock control or H1N1 PR8 infected A549 (p53 +/+ ) cells at different time points (18, 24, 48 hpi) was tested by real-time qpcr using primers specifically designed for H1N1 PR8 strain. (c) The mrna expression of the virus HA (hemagglutinin) gene in mock control or H1N1 PR8 infected A549 (p53 +/+ ) cells at different time points was tested by real-time qpcr using primers specifically designed for H1N1 PR8 strain. (d) Immunofluorescence microscopic images of p53 and H1N1 NS1 protein expression in mock control and H1N1 PR8 infected cells at 48 hpi. Overlay images showing co-localization/co-expression of p53 and NS1. (e) Immunofluorescence microscopic images of phosphor-p53 (Ser15) and H1N1 NS1 protein expression in mock control and H1N1 PR8 infected cells at 48 hpi. Overlay images showing co-localization/co-expression of phosphor-p53 (Ser15) and NS1. (f) Immunofluorescence microscopic images of ERAP1 and H1N1 NS1 protein expression in mock control and H1N1 PR8 infected cells at 48 hpi. Overlay images showing co-localization/co-expression of ERAP1 and NS1. DAPI and Brightfield images confirm the relatively equal number of cells captured in (d-f). Scale bars: 50μm (d-f). 7

8 Supplementary Figure S7. Blocking of IFNβ using neutralizing antibody only partially blocks PR8- induced MHC class I increase in A549 (p53 +/+ ) cells. (a) The IFNγ release from the supernatant of mock control or H1N1 PR8 infected A549 (p53 +/+ ) cells at different time points (18, 24, 48 hpi) was tested by human IFNγ specific ELISA. (b) The IFNβ release from the supernatant of mock control or H1N1 PR8 infected A549 (p53 +/+ ) cells at different time points (18, 24, 48 hpi) was tested by human IFNβ specific ELISA. Data are representative of two independent experiments and error bars represent standard deviation of biological replicates (mean ± s.d., n=3) in a and b. (c) Flow cytometric analysis of MHC class I expression (W6/32 labeling) of H1N1 PR8 (MOI2.0) or mock control infected A549 (p53 +/+ ) cells (Purple) without or with blocking to IFNβ. 1 hour after virus inoculation, the inocula were removed and the cells were maintained in either maintenance media (no blocking) or maintenance media supplemented with different concentrations of IFNβ antibody (1, 5, or 10μg/ml). Cells incubated with fluorescence-labeled secondary antibodies alone served as background controls (Grey). Dotted lines marked the MFI of W6/32 signal observed for A549 (p53 +/+ ) cells in the presence (PR8) or absence (mock control) of virus infection without addition of IFNβ antibody. (d) The calculated MFI for flow cytometric analysis shown in (c). 8

9 Supplementary Figure S8. Full-length images of all immunoblots. 9

10 Supplementary Tables Supplementary Table S1. Primer sequences for generating p53 mutants using site-directed mutagenesis p53 Nucleotide Primer Sequence* (5-3 ) Length (bp) mutants change C176F TGC TTC F : 5'-GGAGGTTGTGAGGCGCTTCCCCCACCA-3' 27 R : 5'-TGGTGGGGGAAGCGCCTCACAACCTCC-3' 27 G245D GGC GAC F : 5'-TCCCGCATGGGCGACATGAACCGGAGG-3' 27 R : 5'-CCTCCGGTTCATGTCGCCCATGCAGGA-3' 27 R273C CGT TGT F : 5'-GAACAGCTTTGAGGTGTGTGTTTGTGCCTGTCCTG-3 35 R : 5'-CAGGACAGGCACAAACACACACCTCAAAGCTGTTC-3 35 R280T AGA ACA F : 5'-TGTGCCTGTCCTGGGACAGACCGGCGCACAGAG-3' 33 R : 5'-CTCTGTGCGCCGGTCTGTCCCAGGACAGGCACA-3' 33 R282W CGG TGG F : 5'-CCTGGGAGAGACTGGCGCACAGAGG-3' 25 R : 5'-CCTCTGTGCGCCAGTCTCTCCCAGG-3' 25 *Light grey background highlights the mutated amino acid and red background highlights the specific nucleotide to change. 10

11 Supplementary Table S2. Primer sequences for cloning of ERAP1 ORF to pcdna3.1 vector ORF Primers Primer Sequence* (5-3 ) Enzyme ERAP1a/b ERAP1-ORF-F1 ACCCAAGCTGGCTAGCATGGTGTTTCTGCCCCTCAA Nhe I ERAP1a/b ERAP1-ORF-R1 TCCATAGCACCAGACTGAAAGTCG ERAP1a/b ERAP1-ORF-F2 GTCTGGTGCTATGGAAAACTGG ERAP1a/b ERAP1-ORF-R2 GGACAGCTTCCCAATGCTGA ERAP1a/b ERAP1-ORF-F3 ATTGGGAAGCTGTCCATTGAAAAG ERAP1a ERAP1-ORF-R3a GCCCTCTAGACTCGAGTCATCCTGTTGCGTCAGCTTC Xho I ERAP1b ERAP1-ORF-R3b GCCCTCTAGACTCGAGTTACATACGTTCAAGCTTTTCACTTTG Xho I *Light grey background highlights the vector homology sequence for In-fusion cloning and dark grey background highlights the enzyme digestion site. 11

12 Supplementary Table S3. Primer sequences for real-time qpcr validation Gene Primers Primer Sequence (5-3 ) Product Length p53 p53-f CCAGGGCAGCTACGGTTTC 205bp p53-r CTCCGTCATGTGCTGTGACTG p21 p21-f GACACCACTGGAGGGTGACT 331bp p21-r GGATTAGGGCTTCCTCTTGG ERAP1 ERAP1-F AGCCCACATGGTAATGGGTA 196bp ERAP1-R TTCACTTTGCAGCCACACTC ERAP2 ERAP2-F TGGGTGAGGAGAAATTCCAG 173bp ERAP2-R GGCGAGCATGTTACTTGTCA PSMB10 PSMB10-F AATGTGGACGCATGTGTGAT 152bp PSMB10-R TCCAGGGTTAGTGGCTTCAC PSMB8 PSMB8-F CACGGGTAGTGGGAACACTT 176bp PSMB8-R TCACCCAACCATCTTCCTTC PSMB9 PSMB9-F ATGCTGACTCGACAGCCTTT 128bp PSMB9-R GCAATAGCGTCTGTGGTGAA PSME1 PSME1-F CAAGGTGGATGTGTTTCGTG 131bp PSME1-R TGCTCAAGTTGGCTTCATTG PSME2 PSME2-F GAAAGTCCTGTCCCTGCTTG 153bp PSME2-R CTCTCCAGCACCTTCTCCTG TAP1 TAP1-F CGTCCACCCTGAGTGATTCT 103bp TAP1-R GACACTGATCCCCAGAGCAT TAP2 TAP2-F AGGAGGCTGCTTCACCTACA 124bp TAP2-R TGAGTTCAGCTCCCCTGTCT TAPBP TAPBP-F CAGTCACCACTGAGCAGCAT 162bp TAPBP-R GCAGAAGAAAGGCAGACAGG TAPBPL TAPBPL-F GATGAAGACTCCCAGGGTCA 129bp TAPBPL-R CTCAGGGATTGGGTCTGTGT HSPA5 HSPA5-F TAGCGTATGGTGCTGCTGTC 117bp HSPA5-R TGACACCTCCCACAGTTTCA CALR CALR-F TCTCAGTTCCGGCAAGTTCT 109bp CALR-R GTTGCTGAAAGGCTCGAAAC CALR3 CALR3-F CTGTGGAGGGGGCTACATTA 104bp CALR3-R ATCCACAAATATCGGGTCCA CANX CANX-F AACCCGAAGGCTGGTTAGAT 142bp CANX-R CATCCAGGAGCTGACTCACA HLA-B HLA-B-F GCGGCTACTACAACCAGAGC 128bp HLA-B-R GATGTAATCCTTGCCGTCGT B2M B2M-F GTGCTCGCGCTACTCTCTCT 143bp B2M-R TCAATGTCGGATGGATGAAA NS1 (PR8) NS1-F CAGCACTCTTGGTCTGGACA 170bp NS1-R TGGACCATTCCCTTGACATT HA (PR8) HA-F TTGCTAAAACCCGGAGACAC 228bp HA-R CCTGACGTATTTTGGGCACT 12

13 Supplementary Table S4. Primer sequences for cloning of putative RE sequences to pgl3- Promoter vector Gene Primers Primer Sequence (5-3 ) Length ERAP1 ERAP1-RE1-F CTCACTTGCCTTAATTTTAGGATACTAGCATTCACTTGCCTT 62bp AATTTTAGGATACTAGCATC ERAP1-RE1-R TCGAGATGCTAGTATCCTAAAATTAAGGCAAGTGAATGCTAGT 70bp ATCCTAAAATTAAGGCAAGTGAGAGCT ERAP1-RE2-F CTGTCATGTGTCAGGCACATGTTATGTCATGTGTCAGGCACA 71bp TGTTATGTCATGTGTCAGGCACATGTTAC ERAP1-RE2-R TCGAGTAACATGTGCCTGACACATGACATAACATGTGCCTGA CACATGACATAACATGTGCCTGACACATGACAGAGCT 79bp 13