Figure 3

Gal-3 Increases RalA activity and Increases YAP1/TEAD and c-MYC transcriptional activity. (A) Active RalA GTP activity was determined in Snu1 and GT-5 Gal-3 cells and control cells (V). Equal amounts of protein from Snu1 and GT-5 control cells (V) or Gal3 overexpressed cells (Gal-3) were incubated with agarose beads coated with RalBP1-RBD, and active RalA-GTP and YAP1 was detected in the complex of RalA and RalBP1 as described in Materials and Methods. YAP1 strongly bind RalA/RalBP1 only in Gal-3 stably transfected GAC cells (Gal3) which is concomitant to increased RalA activity. (B) Correlation expression of Gal-3 and RalA in GAC patients from TCGA database (http://www.cbioportal.org/). (C) Real-time quantitative PCR for Gal-3 and YAP1 and CTGF RNA expression in Snu1 GAC cells with (Gal-3) or Vector (V) stably transfection were performed as described in Materials and methods. (D) YAP1/TEAD, c-MYC and CTGF luciferase promoter activities were determined by co-transfection of Gal4-Tead and 5XUAS-luciferase; c-MYC and CTGF luciferase promoter plasmids respectively. Luciferase reporter activity was measured after 48 h of transfection. For all experiments, values shown represent the mean and s.d. of at least triplicate assays (**P<0.01). (E) YAP1/TEAD transcriptional activity was determined by co-transfection of Gal4-Tead and 5XUAS-luciferase and YAP1 cDNA in Snu1 GAC cells with or without Gal-3 stably transfection; (F). c-MYC luciferase promoter activity was determined by co-transfection c-MYC luciferase promoter construct and YAP1 cDNA in Snu1 GAC cells with or without Gal-3 stably transfection; Luciferase reporter activity was measured after 48 h of transfection. For all experiments, values shown represent the mean and s.d. of at least triplicate assays (*P<0.05; **P<0.01).