Supp Data.Pdf
Supplementary Methods Verhaak sub-classification The Verhaak called tumor subtype was assigned as determined in the Verhaak et al. manuscript, for those TCGA tumors that were included at the time. TCGA data for expression (Affymetrix platform, level 1) and methylation (Infinium platform level 3) were downloaded from the TCGA portal on 09/29/2011. Affymetrix data were processed using R and Biocondutor with a RMA algorithm using quantile normalization and custom CDF. Level 3 methylation data has already been processed and converted to a beta-value. The genes that make up the signature for each of the 4 Verhaak groups (i.e. Proneural, Neural, Mesenchymal, and Classical) were downloaded. For each tumor, the averaged expression of the 4 genes signatures was calculated generating 4 ‘metagene’ scores, one for each subtype, (Colman et al.2010) for every tumor. Then each subtype metagene score was z-score corrected to allow comparison among the 4 metagenes. Finally, for each tumor the subtype with the highest metagene z-score among the four was used to assign subtype. Thus by this method, a tumor is assigned to the group to which it has the strongest expression signature. The shortcoming is that if a tumor has a ‘dual personality’ – for instance has strong expression of both classical and mesenchymal signature genes, there is some arbitrariness to the tumor’s assignment. Western Blot Analysis and cell fractionation Western blot analysis was performed using standard protocols. To determine protein expression we used the following antibodies: TAZ (Sigma and BD Biosciences), YAP (Santa Cruz Biotechnology), CD44 (Cell Signaling), FN1 (BD Biosciences), TEAD1, TEAD2, TEAD3, TEAD4 (Santa Cruz Biotechnology), MST1, p-MST1, MOB1, LATS1, LATS2, 14-3-3-, 1 ACTG2, (Cell Signaling), p-LATS1/2 (Abcam), Flag (Sigma Aldrich), Actin (Calbiochem), CAV2 (BD Biosciences), CTGF (Santa Cruz), RUNX2 (Sigma Aldrich), Cylin A, Cyclin E, Cyclin B1, p-cdk1, p-cdk4 (Cell Signaling Technologies).
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