Verteporfin downregulates YAP/TAZ-TEAD activity and transcriptomes related to adhesion, EMT, and invasion in GBM. (A) Immunocytochemistry analysis of subcellular expression of TAZ, YAP, TEAD1, and the TEAD1-target EGFR after 3-day treatment with Verteporfin (VP) (IC50 dose) vs vehicle (VEH) in G-13063 cells; *P = .017 (YAP), ***P = .0001 (TAZ), **P = 0.0046 (TEAD1), ***P = .00001 (EGFR) by Student t test; n = 24-51 images (dots) in 2-5 wells/condition; lines in box-and-whisker plots represent mean and bars represent min/max. See also Supplementary Figure 2. (B) Representative images of TAZ and TEAD1 from part A. Scale bars = 25 µm. (C) Representative images of EGFR from part A. Scale bars = 50 µm. (D) Volcano plot of differential expression RNA-seq data from 3 biologically distinct GBM lines (G-13063, G-16302, U87) treated with VP (IC50 dose) vs VEH for 3 days. Defined set of 574 downregulated and 164 upregulated genes are marked by blue and red dots, respectively (10% false discovery rate [FDR] adjustment). (E) TEAD-target gene expression in the differential RNA-seq dataset from (D) (*P < .05 and *'P < .1 by Student t test; n = 3 cell lines; bars represent mean ± SEM). (F) Functional enrichment analysis on the differential sets of genes from part (D) (log2-fold change >0.5 and <-0.5) against several gene set databases, HOMER tool. Shown are selected top enriched biological processes, pathways, and reactomes of downregulated (blue) or upregulated (red) VP-associated genes (see Supplementary Data 1 for full list). (G) Gene Set Enrichment Analysis (GSEA) of the predefined signature gene sets “HALLMARK EPITHELIAL MESENCHYMAL TRANSITION” (M5930) and “Multicancer Invasiveness” 33 in the VP/VEH rld normalized RNA-seq dataset. (H) GSEA analysis of rld normalized RNA-seq dataset from CRISPR/Cas9 TEAD1 knockout cell lines vs sham (G-13063, G-16302, G-12746, and G-13306 cell lines8), phenocopying the enrichment pattern seen in VP-treated cells. Abbreviations: EMT, epithelial-mesenchymal transition; GBM, glioblastoma; NES, normalized enrichment score.