Figure 1. Higher expression of YAP1 in NSCLC CSCs.

(A) Elevated YAP1 staining is seen in human lung adenocarcinoma and its metastatic sites as compared to the normal human lung tissue in TMA. SCC, Squamous cell carcinoma; AC, Adenocarcinoma. (B) Quantitation of IHC performed on the TMA shows a 3-fold increase in YAP1 expression in human lung adenocarcinoma tissue. (C) Significant correlation between higher levels of YAP1 and poor prognosis is observed by survival analysis of data from Director’s challenge set. (D) SP cells from A549 and H1650 cells show the ability to self-renew in serial sphere-formation assays in stem cell selective medium. (E) Quantitation of the average number of spheres formed from 1000 cells plated for serial sphere formation assay exhibit higher number of primary and secondary spheres in SP cells as compared to MP cells from A549 and H1650 cells. (F) Aldefluor staining of A549 and H1650 cells shows the presence of Aldh^high population in both A549 and H1650 cells. Aldh activity inhibitor DEAB, is used to gate and separate the Aldh^high cells. (G) Sphere formation assay and its quantitation show the ability of Aldh^high population to form spheres in stem cell selective medium in both A549 and H1650 cells. The numbers of spheres are more in Aldh^high population than the Aldh^low from both A549 and H1650 cells. (H) Western blot analysis on isolated SP and MP cells from NSCLC cell lines for YAP1 protein exhibit higher YAP1 protein levels in A549, H1650 and H1975 SP cells as compared to MP cells. β-Actin is used to show equal amounts of protein in the lanes. (I) qRT-PCR analysis of mRNA from SP and MP cells of A549, H1650 and H1975 cell lines reveal higher levels of YAP1 mRNA in SP cells. ABCG2 mRNA expression is used as positive control for SP cells (J) Elevated levels of YAP1 mRNA seen in sorted Aldh^high cells compared to Aldh^low cells from A549 and H1650. The mRNA expression of Aldehyde dehydrogenase-1 (Aldh1) is used as a positive control.