Fig. 2

Upregulation of GRK3 increased GAC cell growth and invasion, while downregulation of GRK3 inhibited malignant behaviors of tumor cells in vitro. A GRK3 expression levels in normal gastric epithelial cell lines (GES-1 and HFE145), GAC cell lines (AGS, MKN45, GT-5, KATO III, SNU-1, and SNU-16), and patient-derived ascites cells (GA0518, GA0804, GA0515, and GA0313) were determined by western blot. Quantification of fold change for each cell line relative to GES-1 normal gastric cells and β-actin level was performed using Image J. B GRK3 was overexpressed in MKN45 cells by transducing GRK3 cDNA and confirmed by western blot (left) and qPCR (right). C Invasion assay was performed in MKN45-GRK3 overexpression cells compared to that of MKN45-GFP control cells; *** P < 0.0001. D Colony formation assay was performed in MKN45-GRK3 overexpression cells compared to that of MKN45-GFP control cells. E MKN45-GFP control and MKN45-GRK3 OE cells were cultured in media with a series of FBS % for 6 days. Cell survival and proliferation were measured by alamar blue viability assay. x-axis represents the series of FBS % in the culturing media. Values on y-axis are fold changes of growth for each % FBS relative to 0% FBS in either cell line. P values were calculated using the 2-sided Student’s t-test. ***: P < 0.005; **: P < 0.01. F GRK3 was overexpressed in KATO III cells by transfecting wild type (WT; GRK3 OE) or kinase-dead mutant form of GRK3 cDNA and confirmed by western blotting (left) and qPCR (right). G Tumor sphere formation assay was performed in KATO III GRK3 OE or mutant cDNA compared to that of KATO III-GFP control cells and quantification was quantified by image J analyses. *P < 0.01; ** P < 0.001. H GRK3 was KD in GA0518 PC cells using doxycycline inducible system and validated using western blot (left) and qPCR (right). I. GA0518 cells seeded in a 96-well plate were infected with a series of shRNA virus doses of shGRK3 #1 and shGRK3#2 and shScramble, from 20ul to 2.5ul per well. Cell survival and proliferation were measured by alamar blue viability assay 7 days after infection. X-axis represents the series of shRNA virus doses in the infections. Values on Y-axis are relative growth which was normalized to the scramble control shRNA for each virus dose for either cell line. P values were calculated using 2-sided Student’s t-test. ****: P < 0.0001; ***: P < 0.005; *: P < 0.05. J. Colony formation was determined in GA0518 cells with 2 independently GRK3 knockdown clones compared with control. **P < 0.001