Supplementary Information (SI)
SI Materials and Methods
Cell adhesion assay
For the cell adhesion assay, at the end of different treatments, cells (1104) were
trypsinized and re-plated on a 96-well plate pre-coated with type-1-collagen (CN-5
μg/mL), fibronectin (FN, 2 μg/mL), vitronectin (VN, 2 μg/mL), and Matrigel (MG-50
μg/mL). After 2 h, the adhered cells were Hema-3-stained, counted and percentage of
cell-adhesion was represented graphically.
Human tumor samples
Human Glioma Tissue Microarray (GL807; U.S. Biomax Inc, Rockville, MD),
containing glioma tissue samples of different pathological grades and normal brain
tissues (NB) was used in the present study. In addition, normal brain tumor tissues (NB)
and clinically classified human brain tumor biopsies (glioblastoma multiforme WHO
grade IV, GBM) were obtained during autopsies of GBM patients within 24 hours of
death or from patients who underwent surgery at OSF Saint Francis Medical Center,
Peoria, IL (n=30). All samples were collected under protocols approved by the University
of Illinois College of Medicine (Peoria, IL) Institutional Review Board. The nuclear and
cytoplasmic proteins in NB and GBM samples were fractionated using NE-PER Nuclear
and Cytoplasmic Extraction Kit following the manufacturer’s instructions and subjected
to co-immunoprecipitation as described in Materials and Methods.
In vivo subcutaneous tumor experiments
Subcutaneous tumors (S.C.) were developed by injecting 4910 cells (5×106 cells/200 μl
of serum-free medium) in athymic nude mice (n=10). After 10 days, the tumors (>5 mm)
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were injected with pSV and Stat3.si plasmids (300 μg each) on alternate days for a total
of four injections as described earlier (Lakka et al. 2002). After 24 h of first and second
plasmid deliveries, the animals were given radiation treatments (8 Gy) exclusively to the
tumor area by covering the rest of the body with a lead sheet. The tumor size was
measured on every alternate day and total tumor volume was calculated using the formula
1/6 Π (Rmax) × (Rmin)2, where Rmax and Rmin are the maximum and minimum tumor radii
respectively. Animals were sacrificed and S.C. tumors were excised and stored at -80°C
for further molecular analysis. Total RNA from cells and tissues was isolated using
TRIzol reagent and the cDNA was synthesized with RNA (1 µg) and ImProm-II Reverse
Transcription Kit (Promega Corporation, Madison, WI) following the manufacturer’s
protocol. About 1 µl of diluted cDNA (1:10) was used as a template in real-time PCR
using the following parameters: 1 cycle of 95ºC 10 min and 40 cycles of 95ºC 15 s, 60ºC
30 s, 72ºC 30 s, followed by 1 cycle of 72ºC for 10 min. Nuclear and cytosolic proteins
were extracted from S.C. tumors as described above and subjected to Western blotting.
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Supplementary Figure Legends
Supplementary Figure S1 Irradiation (IR) enhanced p-p65/p-Stat3 nuclear localization
in glioma xenograft cell lines 4910 and 5310. (A) IR upregulated p-Stat3, p-p65 and
acetyl-p65 levels in both cell lines. Cells were subjected to IR treatments (0-8 Gy) for 24
h and whole cell lysates were subjected to Western blotting. Representative blots from
three independent experiments were presented. Blots were subsequently stripped and reprobed with GAPDH to confirm equal loading. (B) IR elevated persistent nuclear loc
alization of NF-κB and Stat3. Immunoflorescence experiments show the IR-induced p65
(red) and Stat3 (green) interaction by colocalization (yellow) in 4910 and 5310 at 6 h and
12 h after IR (8 gy) treatment. DAPI was used for nuclear counterstaining and
representative pictures from three individual experiments were presented.
Supplementary Figure S2 Stat3 and NF-κB (p65) interact in the nucleus both in vitro
and in vivo in glioma. (A) Nuclear extracts from U87MG, U251 and SNB19 cells were
subjected to immunoprecipitation (IP) using specific antibodies against p-Stat3 and p-p65
and representative blots were shown. Experiment was repeated thrice to confirm the
results. (B) The nuclear and cytoplasmic proteins in normal brain tissue (NB) and
glioblastoma (GBM) patient tumor biopsies (n=30) were fractionated using NE-PER
Nuclear and Cytoplasmic Extraction Kit following the manufacturer’s instructions.
Nuclear extracts (500 μg) were subjected to co-IP with p-p65, p-Stat3 and IgG antibodies
using μMACSTM protein G microbeads and MACS Separation Columns following the
manufacturer’s instructions (Miltenyi Biotec, Germany). Pre-immunoprecipitated input
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samples (50 µg) were subjected to Western blotting and probed with specific antibody
used for IP to check antibody specificity. (C) The high expression and nuclear
colocalization of NF-κB (p65) and Stat3 in glioma tissues was confirmed by
immunofluorescence experiments using Human Brian Glioma Tissue Array which
comprises normal brain tissue (NB) and glioma tumors of different grades. Tissue array
was subjected to immunofluorescence experiments to analyze the expression levels of pStat3 (red) and p-p65 (green) using HRP-conjugated specific antibodies (1:100 dilution).
Slides were subsequently incubated with respective Alexa Flour® conjugated secondary
antibodies for 1 h at room temperature. Slides were washed and mounted. DAPI was used
for nuclear counterstaining. Negative control staining was performed by incubation with
non-specific IgG (bottom). Insets were enlarged to show 60 × magnifications. (D) The
intensity of p-Stat3/p-p65 nuclear colocalization and the total number of immunopositive
cells were scored from randomly selected microscopic fields. The mean±SE values of
total immunopositivity scores for nuclear Stat3/p65 colocalization in NB, Grade II, Grade
III and Grade IV tumors were plotted in the bar diagram and significance was represented
by *p<0.05, **p<0.01 and ***p<0.001 respectively.
Supplementary Figure S3 The 4910 and 5310 cells were treated with control (0 Gy) and
IR (8 Gy) and cell adhesion experiments were performed as described in SI Materials and
Methods. Percentage of cell adhesion on collagen (CN), vitronectin (VN), fibronectin
(FN) and matrigel (MG) was graphically represented as mean±SE obtained from three
independent experiments (* at p<0.01).
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Supplementary Figure S4 (A) The 4910 and 5310 cells were subjected to different
treatments and ICAM-1 levels were determined using quantitative RT-PCR. Relative
mRNA levels were represented by mean±SE obtained from three individual experiments
and significance was indicated by * at p<0.01. (B) The 4910 and 5310 cells were
subjected to various treatments and EMSA was performed with nuclear extracts (5 µg) to
detect NF-κB DNA-binding activity and representative blots from three independent
experiments were presented. The TNF-α-treated NE samples were incubated with antip65 ab (2 μg) for 30 min before adding the labeled NF-κB probe and loaded to indicate
the antibody supershift.
Supplementary Figure S5 IR-induced invasion and migration in glioma xenograft cells
is mediated by ICAM-1. (A) Matrigel invasion assay was performed and percentage of
cell invasion obtained in different treatments was represented as mean±SE obtained from
three repetitions (* at p<0.01). (B) The relative migration distance values obtained from
wound-healing migration assay after different treatments were represented as mean±SE
obtained from at least three experimental replicates. Significance was denoted by * at
p<0.01.
Supplementary Figure S6 Effect of Stat3.si on IR-induced p65/Stat3 nuclear
localization and ICAM-1 expression in in vivo tumors samples. (A) Real-time
quantitative PCR was performed in subcutaneous (S.C.) tumor biopsies as described
above (n=10). Stat3.si treatment alone significantly inhibited transcription levels of
ICAM-1 when compared to pSV-treated controls. The combination treatment of
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Stat3.si+IR reversed and counteracted the elevated ICAM-1 levels in pSV+IR treated
tumors. (B) Nuclear extracts from pSV, Stat3.si, pSV+IR and Stat3.si+IR treated S.C.
tumors were subjected to Western blotting to determine the expression of p-Stat3 and pp65. HDAC-1 served as loading control. Western blotting was carried out to check
ICAM-1 levels in the whole tumor lysates as described above. Representative blots from
two independent experiments were shown and GAPDH served as loading control. (C) At
the end of the experiment, the total tumor size was measured as described in SI Materials
and Methods and represented as mean±SE obtained from two independent experiments.
Significant difference among different treatments was denoted by * p<0.01.
Supplementary (SI) Reference
Lakka SS, Rajan M, Gondi CS, Yanamandra N, Chandrasekar N, Jasti SL, et al.
Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor
growth and invasion. Oncogene 2002;21: 8011-9.
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