Supplementary Figure Legend
Supplementary Figure 1. IGFBP2-induced STAT3 activation is mediated through
EGFR. Immunoblot analysis of SNB19.BP2 cells depleted of EGFR by using 2
independent pools of EGFR siRNA (EGFR sir#1, EGFR sir#2) or scrambled negative
control siRNA (ctrl siR), followed by overnight serum starvation and stimulation with
100ng/mL recombinant human IL6 (rhIL6) for 15 minutes.
Supplementary Figure 2. Inhibition of ADAMs do not affect IGFBP2-mediated
EGFR signaling activation. (A) U87 glioma cells were serum-starved overnight, then
pretreated with an ADAMs inhibitor, TAPI-2 or marimastat (MMS) (20M), for 2 hours.
After pretreatment, cells were stimulated with 100ng/mL IGFBP2 in serum-free medium
with fresh TAPI-2 or marimastat (20M) for 5 minutes. Whole-cell lysates were collected
for immunoblotting. (B) SNB19.EV and SNB19.BP2 cells were transfected with
ADAM17 siRNA or scramble negative control siRNA for 48 hours and serum-starved
overnight, then treated with 100ng/mL IGFBP2 for 5 minutes. Whole-cell lysates were
collected for immunoblotting.
Supplementary Figure 3. Hierarchical clustering of 157 experimentally validated
STAT3 target genes from Ingenuity Pathway Analysis across all samples in the
Rembrandt glioma dataset. Two distinct clusters formed and associated with tumor
grade and IGFBP2 and STAT3 expression, but not with CTNN1B or FOXM1 expression.
EGFR expression was elevated in a subset of glioblastomas. Blue bar represents low-grade
glioma, and yellow bar represents high-grade glioma.
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Supplementary Figure 4. Migration and invasion potential were significantly
impaired in the SNB19.BP2ΔNLS cells. (A) Diagram of IGFBP2 domains and nuclear
localization signal (NLS). (B) A migration assay was performed on SNB19.EV (empty
vector), SNB19.BP2 WT (wild type) and SNB19.BP2 with a mutant NLS
(SNB19.BP2ΔNLS or mutNLS) cells using a transwell migration chamber. (Left) Cells
were fixed and stained after incubation for 4 hours. (Right) Bar graph represents the mean
number of migrated cells in 5 random view fields (mean ± SD). (C) An invasion assay
was performed on SNB19.EV, SNB19.BP2 WT and SNB19.BP2ΔNLS cells using a
transwell invasion chamber. (Left) Cells were fixed and stained after incubation for 16
hours. (Right) Bar graph represents the mean number of invaded cells in 5 random view
fields (mean ± SD). Indicated annotations correspond to the following P-values: *P<0.05,
***P<0.005, and ****P<0.0001.
Supplementary Figure 5. IGFBP2 promotes nuclear EGFR accumulation in T98G
glioma cells. (A) Immunoblot analysis comparing cytoplasmic and nuclear fractions of
T98G cells depleted of IGFBP2 via 2 independent pools of IGFBP2 siRNA (BP2 siR #1,
#2) for 48 hours to those treated with scrambled negative control siRNA (ctrl siR).
Densitometric analysis of EGFR represented by the bar graph, demonstrates percentage of
cytoplasmic (cyt) or nuclear (nuc) EGFR. (B) Immunoblot analysis of cytoplasmic and
nuclear proteins in T98G cells transiently transfected with EV, BP2 WT and BP2ΔNLS.
Densitometric analysis of EGFR represented by the bar graph, demonstrates percentage of
cytoplasmic (cyt) or nuclear (nuc) EGFR.
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Supplementary Figure 6. Mutation of the IGFBP2 NLS does not affect binding to
EGFR. U87 glioma cells were transiently transfected with EV, BP2 WT or BP2ΔNLS
plasmid followed by immunoprecipitation (IP) for IGFBP2 and immunoblotting (IB).
Supplementary Table 1. Levels of IGFBP2, pSTAT3(Y705) and EGFR in a TMA of
glioma samples from patients were evaluated using immunohistochemical analysis.
Correlation levels were calculated by using the Pearson chi-square analysis.
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