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ANA_23674_sm_SuppFig1.tif1360KSupplementary Figure 1. Validation of laminin alpha chain antibody specificity. The specificity of laminin chain antibodies (Ab) was assessed by evaluating reactivity to a concentration range of purified laminin α2 (0-50ng). An anti-laminin α2 but not anti-laminin α3 or anti-laminin α5 antibody was capable of detecting purified laminin α2 as shown in representative immunoblots.
ANA_23674_sm_SuppFig2.tif8698KSupplementary Figure 2. Tissue microarray analysis confirms elevation of laminin α2 in GBM versus Grades II and III glioma. Representative staining at 10x (left) and 40x (right) of a tissue microarray containing 75 tumor samples shows that laminin α2 is increased in GBM versus lower grade tumors. Expression of laminin α2 was significantly higher (p<0.05) in GBM versus lover grade tumors.
ANA_23674_sm_SuppFig3.tif2560KSupplementary Figure 3. Laminin receptors integrin α6 and β1 are differentially expressed in GBM molecular subtypes. Box plots (D) of association of integrin α6 and integrin β1 expression with molecular subtype in 188 GBMs. Values for gene dosage are presented as log2R/G ratios as estimated by the robust multigene average preprocessing (RMA) algorithm. The box plots show the smallest and largest observations (upper and lower whiskers, respectively), the interquartile range (box), and the median (black line). Data points that are more than 1.5 times the interquartile range lower than the first quartile or 1.5 times the interquartile range higher than the third quartile were considered to be outliers. The p-value was calculated with the use of the Wilcoxon rank-sum test and displayed in Supplementary Table 1.
ANA_23674_sm_SuppFig4.tif7468KSupplementary Figure 4. Tenascin-C is highly expressed in GBM and localized in both the perivascular compartment and throughout the tumor mass. Confocal micrographs of immunostaining analysis from GBM patient specimens demonstrate that Tenascin-C (red) is expressed both in the perivascular niche (indicated by staining with an antibody against CD31, green) and the tumor mass (A). Nuclei counterstained with Hoechst 33342, scale bar = 25 µm. Kaplan-Meier survival plot (B) for glioma patients with differential tumor Tenascin-C expression calculated by NCI REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) bioinformatics database (https://caintegrator.nci.nih.gov/rembrandt/) at a level of two-fold upregulation indicates that Tenascin-C is not informative for patient survival (red line indicates two-fold upregulation, green line indicates two-fold downregulation, and yellow line represents intermediate expression with the number or patients per group indicated in the legend). The log-rank p-value for significance of difference of survival between up-regulated group and intermediate group is 0.73, difference of survival between up-regulated group and down-regulated group is 0.28, and difference of survival between down-regulated group and intermediate group is 0.37.
ANA_23674_sm_SuppFig5.tif2798KSupplementary Figure 5. Laminins produced by non-GSCs in vivo. Summary of quantitative real time PCR results from xenografted GBM tumors 387 enriched based on CD133 expression and analyzed without cell culture expansion demonstrates that GSC marker negative cells (non-GSCs, black bars) have elevated levels of laminin chain transcripts when compared to GSC marker positive cells (GSCs, red bars). Bar graph of quantitative real time PCR results for laminin α2, laminin α4, and laminin α5 demonstrates that non-GSCs consistently contain significantly higher levels of laminin α2, α4, and α5 mRNA. A consistent cycle value of below 35 was not achieved for GSCs enriched based on CD133 for laminin α3 in tumor 387 as indicated on graph; *, p < 0.001 as assessed by one-way ANOVA for all groups.
ANA_23674_sm_SuppFig6.tif1825KSupplementary Figure 6. Validation of laminin α2 knockdown specificity. The specificity of laminin a2 chain knockdown was assessed by evaluating laminin α2, α3, and α5 expression by immunoblotting. To determine additional cross-reactivity and compensation of laminin alpha chains after shRNA knockdown, non-targeting control (NT) or laminin α2 knockdowns (KD1, KD2) were evaluated using antibodies against laminin α2, α3, and α5. Immunoblots demonstrate that laminin α3 and laminin α5 chains were detected in non-GSCs with laminin α2 knockdown. Relative intensity, shown below blots, was calculated by normalization to tubulin levels.
ANA_23674_sm_SuppFig7.tif2288KSupplementary Figure 7. Induction of laminin a2 shRNA attenuates GSC growth and increases Caspase 3/7 activity. Validated laminin α2 shRNAs were used to assess growth and survival of GSCs (derived from tumor 387) and compared to both a non-targeting (NT) and uninfected (no virus) control. GSC growth was compromised upon laminin α2 shRNA mediated knockdown (KD) as shown in ATP based growth assay (A) graphs. Laminin α2 knockdown also resulted in increased cell death as shown by graph of Caspase 3/7 activity (B). For growth assays all statistical assessments were done by one-way ANOVA and comparisons shown by color, black represents comparison to no virus, blue represents comparison to NT, red represents comparison to KD1. ***, p < 0.001 between no virus and NT, &&, p < 0.01 and &&& p < 0.001 between KD1 and no virus or NT, +, p < 0.05, ++, p < 0.01, +++, p < 0.001 between KD2 and no virus, NT, or KD1. For caspase assays, ***, p < 0.001 between indicated groups as assessed by one-way ANOVA.
ANA_23674_sm_SuppFig8.tif6269KSupplementary Figure 8. Increase in laminin a2 expression correlates to higher incidence of edema. Representative MRI images (A) and summary table (B) from patients in the top and bottom quartile of laminin α2 expression demonstrate differences in edema with patients in the laminin α2 high group having a higher incidence of edema.
ANA_23674_sm_SuppTab1.tif2630KSupplementary Table. Summary of association of integrin α6 and integrin β1 with GBM molecular subtypes. Table summarizing number of samples evaluated using TCGA for each GBM molecular subtype (Classical, Mesenchymal, Neural, and Proneural) and p values calculated with the use of the Wilcoxon rank-sum test for integrins α6, and integrin β1 between each subtype.

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