Overexpression of Grp78 induces hepatocellular carcinoma cells invasion. (A), Western blot of protein lysates from mock- and Grp78-transfected SMMC7721 cells, indicating Grp78 expression level. The lower blot was used as loading control. (B), A transwell assay using mcok- and Grp78-transfected cells was performed on inserts pre-coated with ECM gel. Images are representative of the invasion of Grp78 overexpressing cells in three separate experiments performed in triplicate. (C), A chick embryo metastasis assay was performed using mock- and Grp78-transfected cells suspended in PBS. The extent of tumor cell invasion was evaluated by human specific Alu PCR using DNA extracts from chick embryonic lungs and livers. Negative control: DNA extracts from untreated 7-day chick embryonic lungs and livers. Positive control: DNA extracts from human colorectal caricinoma. (D), Western blot analysis showing Grp78 level in Grp78 siRNA and control siRNA transfected Grp78 overexpressing cells. (E) Representative image of a transwell assay using Grp78 siRNA knockdown Grp78 overexpressing cells and control siRNA transfected Grp78 overexpressing cells for further elucidating the role of Grp78 in tumor cells invasion. The experiments were repeated for three times in triplicate.