Supplementary Figure 1

Transcription

1 Supplementary Figure 1 PTEN promotes virus-induced expression of IFNB1 and its downstream genes. (a) Quantitative RT-PCR analysis of IFNB1 mrna (left) and ELISA of IFN-β (right) in HEK 293 cells ( ) transfected for 24 h with empty vector (EV) or plasmid for the expression of PTEN, and then untreated (UT) or transfected for another 8 h with poly(i:c) (1 μg/ml). (b, c) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis of Flag-tagged PTEN and β-actin (below) in HEK293 cells transfected for 24 h with luciferase reporter plasmid for IFN-β (IFN-β-Luc; b) or ISRE (ISRE-Luc; c), plus empty vector or increasing amounts (Wedge: 0, 0.01, 0.05, 0.2 and 1 μg) of plasmid encoding PTEN (below lanes), then left uninfected (UI; left) or infected (right) for 8 h with SeV. (d) Quantitative RT-PCR analysis of IFIT1, ISG15, CXCL10 or

2 RIG-I mrna in HEK 293 cells ( ) transfected for 24 h with empty vector (EV) or plasmid for the expression of PTEN, and then untreated or transfected for another 8 h with SeV; results were normalized to those of the control gene GAPDH and are presented relative to those of untreated cells transfected with empty vector. (e) Luciferase assay (as in Fig. 1b) in HEK293 cells transfected for 24 h with luciferase reporter plasmid for IL-6 (IL-6-Luc; top) or IL-8 (IL-8-Luc; bottom), plus empty vector (EV) or plasmid encoding PTEN, then left untreated (UT) or infected with SeV or treated with IL-1β (0.02 μg/ml) for 10 h. *p < 0.05; **p < 0.01; ***p < (unpaired t-test (a, d, e) or one-way analysis of variant (ANOVA) with post hoc Bonferroni t-test (b, c)). Data are from three independent experiments (a; b, c, top; d, e; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (b, c, bottom).

3 Supplementary Figure 2 PTEN deficiency impairs cellular antiviral responses. (a) Microscopy analyzing VSV replication (left) and immunoblot analysis (right) of VSV-GFP, PTEN and β-actin with the indicated antibodies in HEK293 cells ( ) transfected for 36 h with plasmid encoding non-targeting control sirna (sicontrol) or sirna targeting PTEN (sipten1 or sipten2), then infected for another 24 h with VSV-GFP (MOI = 0.001). (b) Plaque assay of VSV in HEK293 cells ( ) transfected for 24 h with sirna-encoding plasmid as in a and left untreated or transfected for 12 h with poly(i:c), then infected or another 24 h with VSV-GFP (MOI = 0.001). (c) Plaque assay of VSV (left) and quantitative RT-PCR analysis of IFNB1 mrna (right) in 786-O cells stably expressing empty vector (EV) or plasmid encoding wild-type PTEN or PTEN(C124S), and left uninfected or infected for 24 h with VSV-GFP (MOI = 0.01). (d) Cell cytotoxicity assays of PC-3 cells stably expressing empty vector (EV) or plasmid encoding wild-type PTEN or PTEN(C124S), then infected for 24 h with increments of VSV-GFP titers. *p < 0.05; **p < 0.01; ***p < (one-way ANOVA (c) or two-way ANOVA with post hoc Bonferroni t-test (b, d)). Data are from three independent experiments (b, c, d; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (a). BF, bright field.

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5 Supplementary Figure 3 PTEN promotes the induction of IFNB1 independently of Akt pathway. (a) Breeding scheme for the generation of inducible PTEN-mutant mice and PTEN-wild-type mice. (b) Schematic presentation (left) and PCR analysis (right) of the genotype of mice with respect to the CreER transgene and the target Pten gene by using mice tails as PCR templates. P1, P2, P3 and P4 indicated the primers specific for Pten loxp and Cre. (c) Immunoblotting analysis of PTEN expression in mice spleen from PTEN-wild-type mice and PTEN-mutant mice (line 1, 2 and 3). (d) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below lanes) in HEK 293 cells transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, VHR, PTP1B, Cdc25A, SHP1 or SHIP1, and then left uninfected (UI; left) or infected (right) for 10 h with SeV. (e) Luciferase assay (as in Fig. 1b) in HEK 293 cells transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, and untreated (left half) or treated for 2 h with LY (5 μm each), then left uninfected (UI) or infected for 10 h with SeV. (f) Luciferase assay (as in Fig. 1b) in HEK 293 cells transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, plus empty vector (left half) or plasmid encoding Akt-AA (right top) or Myr-Akt (right bottom), and then left uninfected or infected for 10 h with SeV. Akt-AA, the dominant-negative form of Akt, whose phosphorylation sites (Thr308 and Ser473) are replaced by alanine; Myr-Akt, constitutively active Akt with a myristoylation moiety that results in enhanced plasma membrane binding. (g) Luciferase assay (as in Fig. 1b) in MCF-7 cells (left), HeLa cells (middle) and PC-3 cells (right) transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, and untreated (left half) or treated for 2 h with wortmannin (1 μm each), then left uninfected (UI) or infected for 10 h with SeV. (h) Quantitative RT-PCR analysis (left) of IFNB1 and VSV nucleocapsid (VSV-N) mrna and immunoblot analysis (right) of Flag-tagged Myr-Akt, Akt-AA and total GAPDH in the livers of wild-type mice (n = 3) with empty vector (EV), plasmid encoding Myr-Akt or Akt-AA delivered to the livers for 9 h by hydrodynamic tail vein injection, infected intrahepaticly with VSV (1 x 10 7 PFU per mouse), and assessed 6 h after infection. (i) Quantitative RT-PCR analysis (left) of IFNB1 and VSV-N mrna and immunoblot analysis (right) of total Akt, Akt phosphorylation at Ser473 (p-akt(s473)) and total GAPDH in the livers of wild-type mice (n = 3) left untreated (UT) or treated intravenously with wortmannin (0.7 mg/kg) or LY (75 mg/kg) for 0.5 h before intrahepatic infection with VSV (1 x 10 7 PFU per mouse), assessed 6 h after infection. *p < 0.05; **p < 0.01 (one-way ANOVA (d, h, i) or two-way ANOVA with post hoc Bonferroni t-test (e, f, g)). Data are from three independent experiments (d, top; e-g; h, i, left; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (b, right; c; d, bottom; h, i, right).

6 Supplementary Figure 4 PTEN promotes IRF3 but not NF-κB-dependent gene transcription. (a) Quantitative RT-PCR analysis of Isg15, Ifit1, Cxcl10, Ccl5, Cxcl1, Il6, Nfkbia or Nfkb1 mrna in Pten +/+ MEFs and Pten / MEFs left uninfected (UI) or infected for 8 h with SeV. (b) Luciferase assay (as in Fig. 1b) in 293-TLR3 (left), 293 (middle) or 293-TLR4 + MD2 + CD14 + cells (right) transfected for 36 h with a luciferase reporter plasmid for NF-κB promoter (NF-κB-Luc), plus plasmid encoding non-targeting control sirna (sicontrol) or sirna targeting PTEN, left untreated (UT) or treated with poly(i:c) (10 μg/ml) or LPS (10 μg/ml), or transfected with poly(i:c) (1 μg/ml) for 10 h. *p < 0.05; **p < 0.01 (unpaired t-test (a, b)). Data are from three independent experiments (a, b; mean and s.d. of three independent biological replicates per group in each).

7 Supplementary Figure 5 Functional screen for the negative phosphorylation site on IRF3 targeted by PTEN.

8 (a) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below lanes) of IRF3 variants in Irf3 / Irf7 / MEFs ( ) transfected with a luciferase reporter plasmid for IFNB1 promoter (IFN-β-Luc), plus empty vector (EV), or plasmid encoding wild-type IRF3 (WT), IRF3 phospho-mimetic or phospho-defective site mutants, left uninfected (UI) or infected for 10 h with SeV. (b) Immunofluorescence microscopy of Irf3 / Irf7 / MEFs transfected for 20 h with plasmid encoding wild-type IRF3 (WT) or mutant IRF3 with S97D substitution, left uninfected (UI) or infected for 12 h with SeV, stained with antibody to IRF3 (green) and DAPI (blue). Scale bars, 10 μm. (c) Immunoblot analysis (as in Fig. 5d) of HEK293 cells transfected for 24 h with wild-type IRF3 (WT) or mutant IRF3 with S97A substitution, then left uninfected ( ) or infected (+) for 8 h with SeV. (d) Immunoblot analysis of HEK293 cells transfected and infected as in c, followed by nucleus-cytoplasm extraction (and SDS-PAGE of the extracts). (e, f) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below) in Irf3 / Irf7 / MEFs transfected for 24 h with luciferase reporter plasmid for IFNB1 (IFN-β- Luc; e) or IFNA4 (IFN-α4; f) promoter, plus plasmid encoding wild-type IRF7 (WT) or mutant IRF7 with the S112A or S112D substitution (top), then left uninfected (UI) or infected for 8 h with SeV. (g) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below) in Pten +/+ MEFs and Pten / MEFs transfected for 24 h with luciferase reporter plasmid for IFNB1 (IFN-β-Luc), plus plasmid encoding wild-type IRF7 (WT) or mutant IRF7 with the S112A or S112D substitution. **p < 0.01 (two-way ANOVA with post hoc Bonferroni t-test (g)). Data are from three independent experiments (a (top), e-g (top); mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (a (below lanes), c, d, e-g (bottom)).

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10 Supplementary Figure 6 Identification of phosphorylation of IRF3 at Ser97 in vivo. (a) Schematic presentation of IRF3 domain and amino acid sequence with phosphorylation sites identified by mass spectrometry highlighted in red. (b) Tandem mass spectrometry analysis of Ser97-phosphorylated peptide EGLRLAEDRpSK. The observation of fragment ions in the CID product ion spectra was used to localize the phosphorylation site. The presence of the intensive signal at m/z (mass to charge ratio) (doubly charged) corresponding to the neutral loss of phosphoric acid (98 Th, doubly charged) due to gas-phase β-elimination from the doubly charged parent ion at m/z indicated that the peptide was serine (or threonine)-phosphorylated. The difference of 167 Th between b9 2+ and b10 2+ at m/z and respectively, which corresponded to a phosphoserine residue, was observed in the spectrum. This detection established the phosphorylation site as Ser97 in the sequence. (c, d) The phosphorylation sites stoichiometry (phosphorylation intensity versus total intensity) (c) and Commassie blue staining (d) of recombinant IRF3 (fused with two tags SBP (streptavidin-binding peptide) and CBP (calmodulin-binding peptide)) expressed in 293 cells, purified and treated with GST or GST-PTEN, and then separated by SDS-PAGE and mass spectrometry analysis. (e) ELISA of rabbit immune serum (1), antibody flow-through (2), anti-phospho-ser97 antibody (3) or anti-nonphospho-ser97 antibody (4), diluted from 1:500 to 1:10 6 (right margin), with Ser97 phospho-peptide (left half) and its relative non-phospho peptide (right half) coated as an ELISA plate. (f) Immunoblot analysis of IRF3 with anti-phospho-ser97 antibody, plus Ser97-phosphorylated peptide (first row) or its relative non-phosphorylation peptide (second row) in Irf3 / Irf7 / MEFs transfected with empty vector (EV), wild-type IRF3 (WT) and mutant IRF3 with the S97A substitution (top); below (input), immunoblot analysis with antibody to IRF3 and β-actin. (g) Immunoblot analysis of PC-3 cells infected for 0-8 h with SeV, followed by nucleus-cytoplasm extraction (5% of cytoplasmic extracts and 10% of nuclear extracts separated by SDS-PAGE). Data are representative of three independent experiments (a-g).

11 Supplementary Figure 7 In vitro and in vivo phosphatase activity of PTEN. (a) Commassie blue staining of recombinant GST-tagged wild-type PTEN, VHR, PTP1B, Cdc25A and mutant PTEN with the C124S, G129E or G129R substitution expressed in E. coli BL21 (DE3) cells and purified. (b, c) Lipid phosphatase activity assay and serine or threonine phosphatase activity assay of the recombinant proteins as in a, incubated for 2 h with PtdIns-(3,4,5)-P3 (b) or Ser or Thr-phosphorylated peptide (c). (d) In vitro phosphatase assay of GST, recombinant wild-type PTEN, mutant PTEN with the C124S substitution, CIP (Calf

12 intestinal alkaline phosphatase) and λ-ppase (Lambda protein phosphatase) (positive control), incubated for 1 h with recombinant Flag-tagged IRF3 (expressed in HEK293 cells and immunoprecipitated with antibody to Flag). Numbers under lanes indicate IRF3 Ser97 phosphorylation band intensity, normalized to that of total IRF3. (e, f) In vitro phosphatase assay of GST, recombinant wild-type PTEN and CIP, incubated for 1 h with the cell lysates of PC-3 cells left uninfected ( ) or infected for 6 h with SeV (+). (g, h) Immunoblot analysis of IRF3 phosphorylated at Ser97 (p-irf3(s97)) and total IRF3, PTEN and GAPDH (g) and ELISA of IFN-β (h) in PC-3 cells stably expressing empty vector (EV) or plasmid encoding wild-type PTEN and infected for 0-12 h with SeV. Data are from three independent experiments (b, c, h; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (a, d-g).

13 1 Supplementary Table 1. PCR Primers for site-directed mutagenesis kit (TOYOBO). S14A (TCG - GCG) S14D (TCG - GAC) S14A-F S14D-F S14-R GCGCAGCTGGACCTGGGGCAACTGGAGGGC GACCAGCTGGACCTGGGGCAACTGGAGGGC CACCAGCCAGGGCAGGATCCGTGGCTTTGG S30A (AGC - GCC) S30D (AGC - GAC) S30A-F S30D-F S30-R GCCCGCACGCGCTTCCGCATCCCTTGGAAG GACCGCACGCGCTTCCGCATCCCTTGGAAG CTTGTTCACCCAGGCCACGCCCTCCAGTTG S82A (TCT - GCT) S82D (TCT - GAT) S82A-F S82D-F S82-R GCTGCCCTCAACCGCAAAGAAGGGTTGCGT GATGCCCTCAACCGCAAAGAAGGGTTGCGT GCGGAAATTCCTCTTCCAGGTTGGCAGGTC S97A (AGC - GCC) S97D (AGC - GAC) S97A-F S97D-F S97-R GCCAAGGACCCTCACGACCCACATAAAATC GACAAGGACCCTCACGACCCACATAAAATC CCGGTCCTCTGCTAAACGCAACCCTTCTTT S123A (TCT - GCT) S123D (TCT - GAT) S123A-F GCTCCGGACACCAATGGTGGAGGCAGTACT S123D-F GATCCGGACACCAATGGTGGAGGCAGTACT S123-R GGTGTCTGGCTGGGAAAAGTCCCCAACTCC S131A&T132A&S133A&T135A (AGT ACT TCT GAT ACC GCT GCT GCT GAT GCT) S131D&T132D&S133D&T135D (AGT ACT TCT GAT ACC GAT GAT GAT GAT GAT) S131A-F GCTGCTGCTGATGCTCAGGAAGACATTCTGGATGAGTTA 1

14 S131D-F GATGATGATGATGATCAGGAAGACATTCTGGATGAGTTA S131-R GCCTCCACCATTGGTGTCCGGAGAGGTGTCTGG S159A (AGC - GCC) S159D (AGC - GAC) S159A-F GCCCTGGCTGTAGCCCCTGAGCCCTGCCCT S159D-F GACCTGGCTGTAGCCCCTGAGCCCTGCCCT S159-R TGGGGGTCCCGGATCTGGGAGTGGGGCCAA S173A&S175A S173D&S175D (AGC CCC AGC - GCC CCC GCC) (AGC CCC AGC - GAC CCC GAC) S173A-F GCCCCCGCCTTGGACAATCCCACTCCCTTC S173D-F GACCCCGACTTGGACAATCCCACTCCCTTC S173-R CCGCAGGGGCTGAGGGCAGGGCTCAGGGGC S231A (TCC - GCC) S231D (TCC - GAC) S231A-F GCCGAAGTGGGAGACAGGACGCTGCCTGGA S231D-F GACGAAGTGGGAGACAGGACGCTGCCTGGA S231-R CCCCACCAGCCGCAGGCCCTCCGGGCAGGA S303A (AGC - GCC) S303D (AGC - GAC) S303A-F GCCGGGCATGGGCCTGATGGCGAGGTCCCC S303D-F GACGGGCATGGGCCTGATGGCGAGGTCCCC S303-R GTTGGGGAGCAGCTCCTCGCTCACTGCCCA S336A/S339A (AGC GGA CGC TCA - GCC GGA CGC GCA) S336D/S339D (AGC GGA CGC TCA - GAC GGA CGC GAC) S336A-F GCCGGACGCGCACCACGCTATGCCCTCTGGTTC S336D-F GACGGACGCGACCCACGCTATGCCCTCTGGTTC S336-R TCCTTCCGTGAAGGTAATCAGATCTACAATGAA S385A/S386A (TCC TCC - GCC GCC) S385D/S386D (TCC TCC - GAC GAC) 2

15 S385A-F GCCGCCCTGGAGAATACTGTGGACCTGCAC S385D-F GACGACCTGGAGAATACTGTGGACCTGCAC S385-R GGCACCCCCTACCCGGGCCATTTCTACCAA S396A&S398A (TCC AAC AGC - GCC AAC GCC) S396D&S398D (TCC AAC AGC - GAC AAC GAC) S396A-F GCCAACGCCCACCCACTCTCCCTCACCTCC S396D-F GACAACGACCACCCACTCTCCCTCACCTCC S396-R AATGTGCAGGTCCACAGTATTCTCCAGGGA S402A&T404A&S405A (TCC CTC ACC TCC - GCC CTC GCC GCC) S402D&T404D&S405D(TCC CTC ACC TCC - GAC CTC GAC GAC) S402A-F GCCCTCGCCGCCGACCAGTACAAGGCCTACCTGCAG S402D-F GACCTCGACGACGACCAGTACAAGGCCTACCTGCAG S402-R GAGTGGGTGGCTGTTGGAAATGTGCAGGTCCACAGT 2 3

16 3 4 Supplementary Table 2. PCR Primers for Fast Mutagenesis System (TransGen Biotech). T3A-F T3A-R T3D-F T3D-R T32A-F T32A-R T32D-F T32D-R T75A-F T75A-R T75D-F T75D-R T207A-F GAATTCACCATGGGAGCCCCAAAGCCAC CTCCCATGGTGAATTCCTGCAGCCCGG GAATTCACCATGGGAGACCCAAAGCCAC TCTCCCATGGTGAATTCCTGCAGCCCGG GTGAACAAGAGCCGCGCGCG CTTCCG CGCGGCTCTTGTTCACCCAGGCCACG GTGAACAAGA GCCGCGACCGCTTCCGCA GTCGCGGCTCTTGTTCACCCAGGCCACG AAGCCAGACCTGCCAGCCTGGAAGAG CTGGCAGGTCTGGCTTATCCCTCCCG AAGCCAGACCTGCCAGACTGGAAGAGGA TCTGGCAGGTCTGGCTTATCCCTCCCG GTGGGAGTTCGAGGTGGCAGCCTTCTACCGG T207A-R CCACCTCGAACTCCCACTCTTCCCCCGG T207D-F GTGGGAGTTCGAGGTGGACGCCTTCTACCGG T207D-R GTCCACCTCGAACTCCCACTCTTCCCCCGG T219A-F CCAAGTCTTCCAGCAGGCCATCTCCTGCCC T219A-R CCTGCTGGAAGACTTGGCGGCCCCGGT T219D-F CCAAGTCTTCCAGCAGGACATCTCCTGCCC T219D-R TCCTGCTGGAAGACTTGGCGGCCCCGGT T237A-F CGAAGTGGGAGACAGGGCGCTGCCTGGA T237A-R CCCTGTCTCCCACTTCGGACCCCACCAG T237D-F CGAAGTGGGAGACAGGGACCTGCCTGGA 4

17 T237D-R GTCCCTGTCTCCCACTTCGGACCCCACCAG T244A-F GCCTGGATGGCCAGTCGCACTGCCAGACCCT T244A-R CGACTGGCCATCCAGGCAGCGTCCTGTC T244D-F GCCTGGATGGCCAGTCGACCTGCCAGACCCT T244D-R GTCGACTGGCCATCCAGGCAGCGTCCTGTC T253A-F CCCTGGCATGTCCCTGGCAGACAGGGGA T253A-R CCAGGGACATGCCAGGGTCTGGCAGTGT T253D-F CCCTGGCATGTCCCTGGACGACAGGGGA T253D-R GTCCAGGGACATGCCAGGGTCTGGCAGTGT T370A-F GGTCAAGGTTGTGCCCGCG TGCCTCAGGGC T370A-R CGGGCACAACCTTGACCATCACGAGCCTC T370D-F GGTCAAGGTTGTGCCCGACTGCCTCAG GGC T370D-R GTCGGGCACAACCTTGACCATCACGAGCCTC T390A-F CTCCTCCCTGGAGAATG CTGTGGACCT T390A-R CATTCTCCAGGGAGGAGGCACCCCCTAC T390D-F CTCCTCCCTGGAGAATGATGTGGACCTGC T390D-R TCATTCTCCAGGGAGGAGGCACCCCCTAC 5 6 5

18 7 Supplementary Table 3. Primers for mrna Quantification. IFNB1 Forward IFNB1 Reverse GAPDH Forward GAPDH Reverse IFIT1 Forward IFIT1 Reverse ISG15 Forward ISG15 Reverse CXCL10 Forward CXCL10 Reverse RIG-I Forward RIG-I Reverse Ifnb1 Forward Ifnb1 Reverse Gapdh Forward Gapdh Reverse Isg15 Forward Isg15 Reverse Ifit1 Forward Ifit1 Reverse Cxcl10 Forward Cxcl10 Reverse Ccl5 Forward Ccl5 Reverse AGGACAGGATGAACTTTGAC TGATAGACATTAGCCAGGAG ATGACATCAAGAAGGTGGTG CATACCAGGAAATGAGCTTG CCTCCTTGGGTTCGTCTACA GGCTGATATCTGGGTGCCTA GAGAGGCAGCGAACTCATCTT CCAGCATCTTCACCGTCAGG GCTCTACTGAGGTGCTATGTTC GGAGGATGGCAGTGGAAGTC TGATGAATGCCACAACACT CTCCAGTTCCTCCAGATTG CCGAGCAGAGATCTTCAGGAA CCTGCAACCACCACTCATTCT ATGGTGAAGGTCGGTGTGAA CGCTCCTGGAAGATGGTGAT CCTCTGAGCATCCTGGTGAG ACTGGTCTTCGTGGACTTGTT CCAAGTGTTCCAATGCTCCT GGATGGAATTGCCTGCTAGA TCAGGCTCGTCAGTTCTAAGTT GATGGTGGTTAAGTTCGTGCTT AGGACTCTGAGACAGCACAT GCAGTGAGGATGATGGTGAG 6

19 Il6 Forward Il6 Reverse Cxcl1 Forward Cxcl1 Reverse Nfkbia Forward Nfkbia Reverse Nfkb1 Forward Nfkb1 Reverse TTCCATCCAGTTGCCTTCTTG AATTAAGCCTCCGACTTGTGAA GTCATAGCCACACTCAAGAA AGACAGGTGCCATCAGAG GATTGAACCACCATAGACCTA GCGACACAGACCTTAGTT AGACAAGGAGCAGGACAT CCAGCAACATCTTCACATC 8 7