Fig. 4

NF2 deficiency induces GC stemness and Wnt signaling via Bcl-2. A Western blot analysis of NF2 in control and NF2-KO SNU-668 cells. B Sphere-forming assay using control and NF2-KO SNU-668 cells. (left) Representative images of tumorsphere and (right) tumorsphere size (top) and number per well (bottom). Bar = 200 µm. C Low NF2 expression is associated with high histological grade in human GC tissue microarray (TMA) data. P value, chi-square test. D and E Immunohistochemical analysis of β-catenin expression in human GC TMA tissues according to NF2 expression. D Nuclear β-catenin, one-way ANOVA, P value = 0.0144, F = 3.320, (E) Membrane β-catenin, one-way ANOVA, P value = 0.0102, F = 3.552. F and G Microarray and reverse transcriptase-quantitative PCR (RT-qPCR) analysis of relative Bcl-2 mRNA expression in (F) monolayer culture of S1 and S1M cells and (G) monolayer and tumorsphere of S1 cells. H Western blot analysis of Bcl-2 in control and Nf2-KO S1M cells. I TOP-Flash luciferase reporter assay measuring Wnt pathway activity in venetoclax-treated S1M cells. Venetoclax was treated at the indicated concentrations for 24 h. J RT-qPCR analysis confirming Bcl-2 knockdown (KD) efficiency in S1M cells. K TOP-Flash luciferase reporter assay measuring Wnt pathway activity in control and Bcl-2 KD S1M cells. L Expression levels of Wnt-dependent transcription (Lgr5, Cd44, and Axin2) in control and Bcl-2 KD S1M cells. M and N Representative immunofluorescence images displaying Bcl-2 (red) paired with active-β-catenin (green) (M) and Bcl-2 (red) paired with YAP (green) (N) across control, Rasa1-KO, Nf2-KO, and Rasa1/Nf2-double-KO S1 tumorspheres. Bar = 25 µm. O and P Sphere-forming assay using control and Nf2-KO S1 (O) and control and NF2-KO SNU-668 (P) cells. Venetoclax treated at indicated concentrations for 5 days. Student's t-test was used for statistical analysis, unless otherwise specified