Fig. 3

Identification of a novel GRK3 inhibitor LD2 that strongly suppressed the aggressive phenotypes of GAC cells in vitro. A The chemical structure of LD2 was shown. B&C The dose curves of LD2 effects in reducing kinase activities of GRK3 and its closest kinase GRK2. Two types of in vitro kinase assays were carried out to determine IC50 values for LD2 inhibition of GRK3 and GRK2: Invitrogen Z-LYTE and Promega ADP-Glo kinase assay. Plotted on y-axis is the % of remaining kinase activities of GRK3 and GRK2 upon treating with a series of LD2 doses. D LD2 suppressed tumor cell growth in GA0518, GA0804 and AGS cells treated with LD2 at the dosage as indicated. Cell survival was detected by MTS assay at 3 day and 6 day respectively. E& F LD2 suppressed colony formation (E), and cell invasion (F) in a dose-dependent manner. *P < 0.01; **P < 0.001. G Invasion assay was performed in KATO III-overexpression of GRK3 compared to that of KATO III-GFP control cells, and then treated with LD2 in indicated dosage for 48 h. Images of invasion (upper panel) and quantification (bottom panel) were shown. *P < 0.01; ** P < 0.001. H Invasion assay was performed in MKN45-overexpression of GRK3 cells compared to that of MKN45-GFP control cells, and then treated with LD2 in indicated dosage for 48 h. Images of invasion (upper panel) and quantification (bottom panel) were shown. *P < 0.01; ** P < 0.001