PMC

(A) Co-immunoprecipitation (IP) of IGFBP2 and EGFR in SNB19.EV control cells versus SNB19.BP2 cells analyzed by immunoblot (IB). (B) Immunoprecipitation of IGFBP2 in U87 cells starved of serum overnight then stimulated with 2 different doses of IGFBP2 for 30 minutes, analyzed by immunoblotting. (C) Confocal microscopy images of immunofluorescence staining for IGFBP2 (green), EGFR (red) and DAPI (blue) in SNB19.BP2 cells show IGFBP2 and EGFR co-localization; blue arrow = cell membrane; purple arrow = cytoplasm; white arrow = nucleus.

(A) Immunoblot analysis of cytoplasmic (cyt) and nuclear (nuc) fractions of SNB19.EV and SNB19.BP2 cells. Beta-tubulin represents a loading control for the cytoplasmic fraction, and PARP represents a loading control for the nuclear fraction. Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. (B) Confocal images of SNB19 parental cells and SNB19 parental cells stimulated with exogenous IGFBP2 protein (250ng/mL for 30 minutes). Cells were stained for EGFR (red) and the nuclei stained with DAPI (blue). (C) Immunoblot analysis of cytoplasmic and nuclear fractions of SNB19 parental cells stimulated with exogenous IGFBP2 (250ng/mL for indicated times). The graph represents fold-change of cytoplasmic or nuclear IGFBP2 and EGFR calculated from densitometric analysis of the immunoblot bands. (D) Immunoblot analysis comparing cytoplasmic and nuclear fractions of SNB19.BP2 cells depleted of IGFBP2 via 2 independent pools of IGFBP2 siRNA (BP2 siR #1, #2) to cells transfected with scrambled negative control siRNA (ctrl siR). Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. (E) Immunoblot analysis of cytoplasmic and nuclear fractions of transiently transfected SNB19.EV, SNB19.BP2 wild type (BP2 WT) and SNB19 with a mutated IGFBP2 nuclear localization signal (BP2ΔNLS). Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. (F) Immunoblot analysis of cytoplasmic and nuclear proteins in stable SNB19.EV, SNB19.BP2 WT and SNB19.BP2ΔNLS cells.

(A) GSEA demonstrated enrichment for STAT3 target genes based on correlation with IGFBP2 expression in the TCGA low-grade glioma database. The top of the panel shows the enrichment score (ES) for genes associated with STAT3 signaling pathway targets. The blue lines indicate where the STAT3 target genes appear in the ranked gene list, and the black lines represent the top 45 highly correlated targets. The bottom of the panel shows the ranking scores (correlation of all genes associated with the STAT3 signaling pathway targets with IGFBP2). (B) Correlation of expression of proteins in the TCGA RPPA data with IGFBP2 (x-axis) and pSTAT3(Y705) (y-axis). Each dot represents a protein. Proteins with correlation coefficients greater than 0.2 are highlighted in orange. (C) Correlation of the 7 proteins with the highest correlation coefficients with both IGFBP2 and STAT3. Also shown is the relationship of each protein with STAT3 (“target” = STAT3 transcriptional target; “regulator” = STAT3 upstream regulator). Y = yes, a known target or upstream regulator of STAT3; N = not a known target or upstream regulator of STAT3.

(A) Immunoblot analysis of SNB19 cells starved of serum overnight then stimulated with exogenous IGFBP2 protein at the indicated dosages (0, 50, 100, 250 ng/mL) for 60 minutes. Densitometric analysis shown below the immunoblot indicates fold-change relative to unstimulated control cells (normalized to beta-actin loading control or total protein for phosphorylated proteins). (B) Immunoblot analysis of U87 cells starved of serum overnight then stimulated with exogenous IGFBP2 (100ng/mL) for the indicated time points (0, 5, 10, 15, 30, 60 minutes). Densitometric analysis shown below the immunoblot indicates fold-change relative to unstimulated control cells (normalized to loading control or total protein for phosphorylated proteins). (C) Immunoblot analysis comparing stable SNB19 empty vector cells (SNB19.EV) to SNB19 cells stably overexpressing IGFBP2 (SNB19.BP2). Densitometric analysis shown below the immunoblot indicates fold-change relative to SNB19.EV after normalization to beta-tubulin loading control (or total protein for phosphorylated proteins). (D) Immunoblot analysis comparing SNB19.EV and SNB19.BP2 cells depleted of EGFR via 2 independent pools of EGFR siRNA (EGFR sir#1, EGFR sir#2) to cells transfected with scrambled negative control siRNA (ctrl siR). The intensity of pSTAT3(Y705), quantified by densitometry, is shown below the immunoblot as fold-change relative to control siRNA, normalized to total STAT3.

Expression and localization of IGFBP2, pSTAT3(Y705) and EGFR were detected with immunohistochemistry from a TMA that included 222 human grade 2-4 gliomas. (A) TMA immunostaining images (magnification 40×) representing weak and strong staining of IGFBP2 and pSTAT3(Y705). (B) Cytosolic and nuclear IGFBP2 expression associated with the percentage of cells positive for pSTAT3 and with pSTAT3 staining intensity. Bar graphs illustrate the increasing fractions of pSTAT3-positive cells and pSTAT3 intensity upon increasing IGFBP2 intensity or nuclear accumulation. (C) TMA immunostaining images (magnification 40×) representing low and high nuclear localization of IGFBP2 and EGFR. (D) Nuclear IGFBP2 associated with nuclear EGFR. The bar graph illustrates the fraction of samples with increasing nuclear EGFR localization upon increasing nuclear accumulation of IGFBP2. (E) Nuclear co-localization of IGFBP2, EGFR and phosphorylated STAT3 predicted poor survival among patients with human grade 2-4 glioma. Patients were stratified into 2 cohorts based on the nuclear staining of all 3 proteins: triple positives (≥1% of cells with nuclear expression, n=51, red line) and all other cases (n=83, blue line). Survival rates were visualized by using a Kaplan-Meier survival plot (p=0.0086).