Expression analyses of EREG and HB-EGF in IRE1α-deficient cells. Analyses were performed using either the dominant-negative strategy (U87dn vs. U87Ctrl cells) or the siRNA IRE1α knockdown methodology. (a) Fold-increase in gene expression was examined from microarray data (GEO, #GSE22385) and by qPCR. For knockdown analysis, IRE1α siRNA-transfected U87wt cells were compared to nontargeting siRNA-treated cells (si.Ctrl). SPARC and THBS1 mRNA levels were given for comparison. qPCR mean values were ± SD. (b) EREG protein levels in cell-conditioned media as determined by ELISA. Results are mean values ± SD. The dotted line represents the limit of detection of the measure. (c) The chicken egg model. Cells were deposited onto the chicken CAM and tumors were allowed to grow for 4 days. Upper panel: microphotographs of U87Ctrl- and U87dn-derived tumors at day 4. Bar = 2 mm. Lower panel: variation of EREG and HB-EGF transcripts levels in U87dn vs. U87Ctrl tumors as measured by qPCR. Data are mean values of five pooled tumors ± SD. (d) Mouse model. Cells were intracranially implanted into the left frontal lobe and tumors were collected at d28 (U87Ctrl) and at d43 (U87dn) post-implantation. Brain sections were stained with H&E (i, iv). Aspect of tumors before (ii, v) and after (iii, vi) LCM (Bars = 300 μm). Tumor areas were dissected inside the tumor core in control animals (ii, iii) and multiple tumor cell bundles were collected in infiltrative dn tumors (v, vi). Gene expression analyses (vii) were carried out by qPCR using HPRT1 as reference. Results are fold-increase ± SD of triplicates in three independent experiments (Exp. 1–3). NC, no change; → 0, No Ct value obtained with U87dn tumors; → ∞, value > 3 000; ND, value could not be determined. Visualization of amplicons after 40 cycles of qPCR (panel vii, right).