Fig. 3

Cooperative enhancement of Wnt and YAP signaling in cancer stem cells by NF2 and RASA1 deficiency. A Western blot analysis of RASA1 and NF2 in control and KO S1M cells. B-F (B) Bioluminescence in vivo imaging of NOD-SCID mice 10 days after intraperitoneal transplantation of control, Nf2-, Rasa1- and Nf2/Rasa1-double-KO S1M cells (each of n = 4). (C) Total bioluminescence signals measured in each mouse. D Representative gross and (E) H&E images of each group. Red dashed lines indicate tumor area. Bar = (D) 1 cm, (E) 200 μm. F Effect of Rasa1- and Nf2-deficiency on peritoneal dissemination was evaluated using ascites volume (top) and number of macro-metastatic foci (bottom). G and H Sphere-forming assay using control, Rasa-, Nf2-, and Rasa1/Nf2-double-KO S1 cells. G Primary and (H) secondary tumorspheres (top) size and (bottom) number was measured. I Principal component analysis of RNA profiles obtained from control and KO S1 tumorspheres. Each dot represents a technical replication. J and K (J) GO and (K) KEGG pathway enrichment analysis of genes with significant expression changes (> threefold change) in KO cells compared to control cells. L Bar chart showing the differential activation of the Wnt and Hippo signaling pathways in KO cells compared to control cells, as determined by KEGG pathway analysis. M mRNA expression levels of YAP/TAZ-TEAD target genes in control, Rasa1-KO, Nf2-KO, and Rasa1/Nf2-double-KO S1 tumorspheres, as determined by RNA sequencing analysis. N Gene set enrichment analysis showing upregulation of Wnt and Hippo/YAP pathway-related genes in double-KO S1 tumorspheres compared to Rasa1-KO tumorspheres. O and P TOP-Flash luciferase reporter assay in control and KO S1M cells, depending on WNT3A stimulation; (O) conventional media, (P) 25 ng/ml of WNT3A. Q Relative mRNA expression of Wnt-dependent transcription (Aqp5, Axin2, and Ccnd1) in control and KO S1M cells (WNT3A, 25 ng/ml). R and S (R) HOP-Flash luciferase reporter assay and (S) relative mRNA expression of YAP-dependent transcription (Ctgf and Cyr61) in control, Rasa1-, Nf2-, and Rasa1/Nf2-double-KO S1 cells. T and U Immunofluorescence staining images of control and KO S1 tumorspheres using (T) active-β-catenin (green) and (U) YAP (green) with DAPI. Bar = 25 μm. V Western blot analysis of MYC, Cyclin D1 and Survivin in control and KO S1M cells (WNT3A, 25 ng/ml). Student's t-test was used for statistical analysis, unless otherwise specified