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1 Supporting Information Sen et al /pnas SI Materials and Methods Cell Culture. Human embryonic lung fibroblasts (HELF) were maintained in Eagle s minimum essential medium (Mediatech) supplemented with 10% FCS (Gemini Bio-Products), amphotericin (0.5 mg/ml; Omega Scientific), and penicillin G and streptomycin (100 U/mL of each; Omega Scientific). Melanoma cells were propagated in this medium supplemented with nonessential amino acids (100 μm; Omega Scientific). Primary tonsil T cells were maintained in RPMI-1640 supplemented with 10% FCS and antibiotics. Immunohistochemistry and Immunofluorescence. Paraffin-embedded skin sections were deparaffinized and rehydrated followed by antigen retrieval. The sections were probed for VZV infection using anti-ie63 antibody (kind gift from William T. Ruyechan, University at Buffalo), total STAT3, and STAT3-Y705 (D3A7) (Cell Signaling Technology). The sections were stained using 3,3 - diaminobenzidine substrate (Vector Laboratories) and counterstained with hematoxylin. For immunofluorescence, deparaffinized tissue sections were treated for antigen retrieval and probed with indicated primary antibodies. The primary antibodies were detected using donkey anti-rabbit Alexa Fluor 488 and goat antimouse Alexa Fluor 594 (Invitrogen). Images were captured using the Leica SP2 confocal microscope (Stanford Cell Sciences Imaging Facility Stanford University). Immunoblots. Whole-cell lysates of infected and uninfected HELF were prepared by lysing cells in 2 Laemmli Sample Buffer (Bio-Rad) containing 5% β-mercaptoethanol (GIBCO and Invitrogen). The lysed samples were boiled for 5 min and separated on a 7.5% or 4 20% gradient SDS/PAGE. Immunoblotting was done using primary antibodies to STAT3, STAT3-Y705 (D3A7), survivin, IκBα (Cell Signaling Technology), and α-tubulin (Sigma). Antibodies to varicella-zoster virus (VZV) IE62 and ORF4 were generous gifts from William T. Ruyechan (University of Buffalo, Buffalo, NY) and Paul R. Kinchington (University of Pittsburgh, Pittsburgh, PA). ORF11 antibody was generated as described previously (1). Horseradish peroxidaseconjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Amersham Biosciences. Plaque Size Quantification. Each plaque was photographed using the Zeiss Axio Scope A1 (5 objective), and size was determined using the ImageJ analysis software (2). Plaque size of the indicated number of plaques from each group was calculated and plotted on a bar chart. Infection of Human T Cells. Human tonsil tissue was obtained according to a protocol approved by the Stanford University Committee for Research Involving Human Subjects. The tonsils were dissociated to single cells and purified by nylon wool column as described previously (3) followed by negative selection using the EasySep human T-cell enrichment kit (STEMCELL Technologies). Infection of T cells with VZV was achieved as previously described (4); T cells were cocultured with a monolayer of roka-infected HELF for 24 h and recovered for flow cytometry and other analyses. Quantitative PCR. For quantitative PCR analyses, cells were lysed and RNA was prepared using TRIzol (Invitrogen). Reverse transcription was carried out at 50 C for 1 h using SuperScript III (Invitrogen), and the cdna was analyzed using the Brilliant II Fast SYBR green kit (Stratagene) as per the manufacturer s protocol in a real-time PCR instrument (Mx3005P; Agilent). Data were collected and analyzed by the comparative quantification method using the instrument software Microsoft Excel spreadsheet software and Prism statistical analysis software (GraphPad). 1. Che X, et al. (2011) Identification and functional characterization of the Varicella zoster virus ORF11 gene product. Virology 412(1):156e Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics International 11:36e Ku CC, Padilla JA, Grose C, Butcher EC, Arvin AM (2002) Tropism of varicella-zoster virus for human tonsillar CD4(+) T lymphocytes that express activation, memory, and skin homing markers. J Virol 76:11425e Zerboni L, Sommer M, Ware CF, Arvin AM (2000) Varicella-zoster virus infection of a human CD4-positive T-cell line. Virology 270:278e285. 1of5

2 Fig. S1. VZV infection directs STAT3 phosphorylation in HELF and T cells. (A) HELF infected with VZV-GFP for 24 h or left uninfected were analyzed by flow cytometry for phosphorylated STAT3 (pstat3) expression in VZV+ (GFP+) and VZV (GFP ) populations. (B) The fluorescence intensity in the VZV+ and VZV populations detected using the Alexa Fluor 647-pSTAT3-Y705 antibody is shown in the histogram. Uninfected HELF and HELF treated with IFNα served as negative and positive controls, respectively. (C) T cells purified from human tonsil tissue were cocultured with VZV-GFP infected HELF and analyzed by flow cytometry for pstat3 expression (24 hours post inoculation) using PE-CD3 and Alexa Fluor 647-pSTAT3-Y705 antibodies. The fluorescence intensity in the VZV+ and VZV populations is shown in the histogram; mock-infected T cells served as the negative control, and IFNα-treated T cells served as the positive control. Fig. S2. HELF treated with DMSO do not alter VZV infection and spread. Immunofluorescence detection of the spread of VZV-GFP in untreated and DMSOtreated HELF at 48 hours post inoculation. 2of5

3 Fig. S3. Inhibition of STAT3 phosphorylation restricts VZV infection of human tonsil T cells. Purified tonsil T cells were cocultured with VZV-infected HELF for 24 h following which the VZV-infected T cells were removed from HELF and treated with either DMSO or 100 μm S3I-201. After a 24-h treatment with DMSO and S3I-201, the T cells were gated on CD3+ and analyzed by flow cytometry for GFP expression (an indicator of virus infection). Fig. S4. Skin xenografts treated with S3I-201 have no changes in tissue morphology or total STAT3 expression. Hematoxylin eosin staining of a section of uninfected skin xenograft from mouse #8 treated with S3I-201 and immunoblot analysis of total STAT3 from DMSO- and S3I-201-treated skin xenografts are shown. 3of5

4 Fig. S5. VZV infection leads to up-regulation of survivin transcription and translation in vitro and in vivo. (A) Immunoblot detection of pstat3-y705 expression in HELF infected with increasing concentrations of VZV inoculum. IL6-treated HELF served as the positive control, and untreated and mock-infected HELF served as the negative control. (B) Survivin transcription profile in infected, IL6-treated, and control HELF was determined by quantitative PCR at 24 hours post inoculation using the SYBR green method. (C) Glycoprotein E (ge)-positive cells in the area marked C show staining for survivin within the cytoplasm of infected cells whereas ge-positive cells in the area marked N show nuclear localization of survivin. (D) Nuclear survivin (Alexa Fluor 488, green) is observed in cells that do not exhibit ge staining (Alexa Fluor 594, red) but are in close proximity to infected cells. Fig. S6. Proposed model for the role of STAT3 and survivin in pathogenesis of VZV in skin. VZV infection results in STAT3 phosphorylation, which ( a in model) by modulating survivin expression regulates apoptosis favoring virus replication and ( b in model) antagonizes pstat1 activation in response to IFNα and therefore overcomes the intrinsic antiviral response of skin cells. 4of5

5 Table S1. Oligo sequences of pstat3 target genes used in the study Target gene Oligo sequence (5 3 ) Amplicon size (bp) Survivin GCA TGG GTG CCC CGA CGT TG 446 GCT CCG GCC AGA GGC CTC AA Bcl-xl GGA AAG CGT AGA CAA GGA GAT GC 236 GGT GGG AGG GTA GAG TGG ATG GT Cyclin D1 GAG ACC ATC CCC CTG ACG GC 484 CTC TTC CTC CTC CTC GGC GGC STAT3 GGG TGG AGA AGG ACA TCA GCG GTA A 298 GCC GAC AAT ACT TTC CGA ATG C Leukemia inhibitory factor CTG TTG GTT CTG CAC TGG A 350 GGG TTG AGG ATC TTC TGG T 5of5