Fig. 1
CD98 is involved in the spreading and tumorigenicity of SMMC-7721 cells in vitro and in vivo. a SMMC-7721 cells were transfected with pLKO–SLC3A2(CD98) shRNA (SMMC-7721shCD98) or pLKO–control shRNA plasmid (control) and selected with 5 μg/ml of puromycin for two weeks. The cells (5 × 104 cells) were grown on coverslips for 24 h, fixed and stained with Dylight594-conjugated goat-anti-mouse antibodies (CD98, Red). Representative CD98 immunofluorescence of control and SMMC-7721shCD98 cells are shown in (a) DAPI (blue staining). Bar, 10 μm. b FCAS analysis of membrane CD98 in control and SMMC-7721shCD98 cells. Growing cells were harvested and incubated with fluorescence-labeled antibody against CD98 (PE) for 1 h. Next, the labeled cells were analyzed using flow cytometry. Mean fluorescence intensities (MFI) of each group was indicated beside the histograms.The data shown are representative of three individual experiments. c SMMC-7721 cell spreading assay in vitro. Control or SMMC-7721shCD98 cells (5× 104 cells) were cultured on coverslips with 1 % matrigel for 2 h, and then the cells were fixed and stained with rhodamine-phalloidin for visualization (right) and scoring (left). The percentage of spread cells was determined by scoring >100 cells. The data are the mean of the “% of spreaded cells” from three independent experiments ± SED. Bar, 10 μm. ** P < 0.01. d Tumorigenicity of cells transplanted into nude mice. Control or SMMC-7721shCD98 cells (5 × 105 cells per site) were injected into both sides of 6-weeks-old female nude mice. Tumor volumes were determined at various time points (see Materials and Methods for details). On day 18, the primary tumors were harvested and weighed (e). *** P < 0.001