Download Supplementary Figure Legends Fig S1: RT

Supplementary Figure Legends
Fig S1: RT-PCR products amplified from different GBM samples shown in Figure 1B were sequenced and
aligned with published sequences for the laboratory strain of HCMV- IE1. Sequence alignment
demonstrates that each gene product isolated in endogenously infected human GBM tissues is distinct
from the IE1 sequence found in the laboratory strain AD169 (published sequence is shown). Each
sequence is shown in a different color, to reflect distinct PCR products.
Fig S2. HCMV IE is enriched in the SSEA1+ fraction of primary GBM cells and co-localizes with stemness
markers in situ. A. IE1 Taqman analysis of stage specific embryonic antigen 1 (SSEA1) cellular fractions
isolated from two different GBM samples. Results are shown normalized to Rab14 values. B-E. Primary
GBM cells isolated from CPMC-100, cultured on laminin were co- stained for IE1(green) and Nestin(A,
bar=100m ), PDGFR(B),Olig 2(C, bar=50m), and Integrin 6 (D), all in red fluorescence. Nuclei,
counterstained with DAPI (A-B) or propidium iodide (C-D).
Fig S3. IE siRNA Optimization (in conjunction with Figure 2A). A. GSC4121 infected with HCMV (TR, 72h,
MOI=1) were treated with non-targeting control siRNA or the combination of IE targeting siRNA
duplexes; photomicrographs illustrate representative examples from each treatment category (bar
=100m). The right panel shows an example of mock infected GSC treated with IE siRNA for 72h,
demonstrating lack of IE siRNA toxicity in uninfected cells. B. Tumorsphere formation assays carried out
in the indicated conditions were quantified 7days following initial culturing. Each condition was run in
quadruplicate wells and the experiment was repeated twice *p<0.02, ** p=0.017, student t-Test. Bars,
SD.
Fig S4. HCMV transcripts are enriched in the GSC fraction. A. GSC 4121 and 0609 were maintained in
nude mice (1).To generate HCMV+ cultures, tumors were dissociated, infected with HCMV (TR or Towne
, MOI=1) and 48h later sorted for the indicated markers. 1ug RNA from each fraction was used for
Taqman detection of the indicated HCMV gene products. Relative abundance was calculated after
normalization to Rab 14. B-C. Limiting dilution assays were carried out for 4121 and 0609 cells using 10,
100, and 1000 cells/well. siRNA duplexes targeting IE1(exon 4) or IE (exon 3) were tested separately and
combined and the number of wells in each well was recorded at seven days. D. Estimated stem cell
frequencies for each condition and the plots were generated using ELDA software available at
http://bioinf.wehi.edu.au/software/elda/.
Fig S5 (In conjunction with Figure 2D -F). HCMV IE modulates Sox2 expression in GBM via miR145. A.
Primary, endogenously HCMV-infected CPMC-085-derived cells were treated with control or IE siRNA
(72h) and harvested in lysis buffer to be hybridized with pluripotent stem cell antibody arrays, according
to the manufacturer’s instructions. 1- represents the location of Sox2 on the array. B. A portion of the
cell lysates from A was used for western blot analysis; this array validation experiment demonstrates
inhibition of Sox2 levels in the IEKD sample. C. GSC3832 (initially HCMV negative) were infected with
mock or TR and treated with control or IE siRNA. 48 h following transfection, cell lysates were hybridized
to stem cell arrays as shown in A. Densitometry measurements were performed, per manufacturer’s
instructions. Graph shows relative expression levels for each transcription factor shown on the X axis in
the indicated conditions. HCMV infection induced upregulation of stemness factors and this effect was
partially reverted by IEKD. C. GSC 3832 were transfected with anti-miR145 (“antagomir”), infected with
HCMV (TR, 1MOI), or pre-treated with mature mimic miR145 and then infected with mock or HCMV.
Forty eight hours following infection, cells were lysed and analyzed by Western blot for the indicated
proteins. E. Sphere formation assays were carried out in three GSC lines (Figure 2G). Here, we show
representative photomicrographs of GSC4121 treated as indicated, 72h following HCMV infection. Bar=
100m.
Fig S6. IE1 overexpression induces Sox2 via miRNA145 inhibition in GSCs. A. Sox2 levels were
measured in LXSN (control), IE1 transduced and HCMV (Towne, MOI=1, 48h) or mock infected GSCs
using western blot. B. Taqman detection of miRNA145 in mock, LXSN, IE1, or HCMV infected GSCs. Bars,
SD. *,p<0.05, Mock vs IE1. **p<0.01, student t-Test. C. Tumorsphere assays were carried out for LXSN,
IE1-expressing or HCMV infected GSC 4121, plated at 10, 100, 1000 cells/well, in quadruplicate.
Representative pics from cultures with 100cells/well, 96h after initial culturing. The experiment was
repeated twice. Bar=75m.
Fig S7 (In conjunction with Figure 5) IE attenuation induces a mesenchymal and pro-inflammatory
phenotype in HCMV-positive GSC. A. A portion of the RNA used for CMV gene profiling shown in Fig 5C
was hybridized to Affymetrix human DNA arrays. Hierarchical clustering shows cellular transcripts
significantly up (red)- or downregulated (blue) by IEKD. B. IPA analysis of data (shown in D) depicts
interactions and pathway connections among a subset of cellular genes significantly altered by IEKD.
Green, downregulated, Red upregulated.
Fig S8 Immunohistochemical analyses of IE1+/- mouse gliomas. A. Low magnification
photomicrographs of spontaneous gliomas +/- IE1 stained for H&E. B. Pathology evaluation of
spontaneous mouse GBMs shows distribution of tumor grade in presence or absence of IE1 (see also
Table S3).C. Representative photomicrographs of mouse glioma samples processed for
immunohistochemistry for the indicated antibodies. Bottom row displays tissue samples from IE1expressing mouse tumors. Sox2 and Ki67 markers were significantly upregulated in the IE1+ gliomas (see
also Fig 6E). Bar=200m.
Fig S9. GSC lines used in this study are tumorigenic in vivo. H& E staining of representative mouse GSCderived xenografts. A-B. Intracranial implantation of 100,000 cells from GSC 4121 resulted in a high
grade GBM 90 days following tumor implantation. C-D. 10,000 GSC0609 cells induced a highly aggressive
GBM 30 days post implantation. Bar= 200um. GSC3832 obtained from Dr. J. Rich laboratory has been
previously published to be highly tumorigenic in vivo (see also Fig S10).
Fig S10. CR208, an IE1 deficient HCMV does not promote GBM stemness to the same extent as Towne
parental strain. A. Western blot analyses of GSC3832 treating as indicated and using antibodies specific
for HCMV IE1/IE2 proteins and Sox2. Actin used as loading control. B. Quantification of 3832 GSC
tumorspheres treated with mock, Towne and CR208 viruses. ** p<0.02, student t-Test. Bars, SD. C-E.
Immunohistochemical analyses of mouse glioma tissues from the mock, Towne or CR208 infected
intracranial xenografts. Tissues were harvested thirty days post tumor implantation. F. Luminescence
measurements from10 mice/group at day 25 post tumor implantation (1000 cells/mouse). Statistical
analysis for luminescence data using ANOVA shows significant differences across the three groups,
p=0.0007. G. Survival data for intracranial tumor-bearing mice. Each group consisted of 10 mice
(experiment was repeated twice).