Fig. 6

Effects of RASA1 deficiency on stemness via Bcl-xL. A (left) Representative images of tumorspheres of control (top) and RASA1-KO (bottom) SNU-484 cells. (right) Tumorsphere size (top) and counts (bottom). Bar = 200 µm. B Low RASA1 expression has correlation with high histological grade human gastric cancer (GC) in tissue microarray (TMA) tissue. P value, chi-square test. C Representative RASA1 immunohistochemical images of human GC TMA according to tumor cell differentiation. D Representative images of Tp53-KO mouse stomach organoids of control and Rasa1-KO. (top) Diameter of organoids and (bottom) number of organoids per well. Bar = 200 µm. E Relative mRNA expression levels of stem cell markers (Lgr5, Cd44, and Axin2) in control and Rasa1-KO mouse stomach organoids. F Representative immunofluorescence staining images for AQP5 (green), PCNA (red), and DAPI in control and Rasa1-KO Tp53-KO organoids represented in (D). Bar = 50 µm. G Western blot analysis of p-ERK, ERK, and Bcl-xL in control and Rasa1-KO S1M cells. H Bcl-xL mRNA expression in S1M cells treated with trametinib (60 nM) for 48 h. I Bcl-xL mRNA expression levels depending on RASA1 mutation status in TCGA GC dataset. J and K TOP-Flash luciferase reporter assay measuring Wnt pathway activity in control and Rasa1-KO S1M cells treated with (J) BH3I-1 and (K) venetoclax at the indicated concentrations for 24 h. L RT-qPCR analysis to confirm knockdown (KD) efficiency of Bcl-xL in S1M cells treated with si-Bcl-xL. M TOP-Flash luciferase reporter assay in control and Bcl-xL KD S1M cells. N RT-qPCR analysis measuring tetracycline induced Bcl-xL expression in S1M cells after doxycycline (Dox) treatment at multiple doses. O TOP-Flash luciferase reporter assay in tetracycline-induced Bcl-xL-overexpression S1M cells. P and Q Representative immunofluorescence images displaying Bcl-xL (red) paired with active-β-catenin (green) (P) and Bcl-xL (red) paired with YAP (green) (Q) across control, Rasa1-KO, Nf2-KO, and Rasa1/Nf2-double-KO S1 tumorspheres. Bar = 25 µm. Student's t-test was used for statistical analysis, unless otherwise specified