scFv validation by HepG2 and HepG2-shRNA cell lines. A. Screening of shRNAs for hGPC3 silencing. HEK 293 cells were transfected with shRNA-harboring pSIREN-ZsGreen vector and hGPC3368-551-expressing plasmid. Expression of myc-tagged hGPC3368-551 was assessed by Western blot using anti-myc antibody. GAPDH was used as control. B-C. Lower expression of hGPC3 in HepG2-sh57 expressing cells. Stable sh57 expression was established in HepG2 cell line via retroviral transduction. hGPC3 expression in these cells was detected by FACS using anti-GPC3 antibody (1G12) followed by anti-mouse APC. Unstained HepG2 cells (grey area), mouse isotype control-stained HepG2 (black line), HepG2 cells (green line), and HepG2-sh57 (red line) are shown. The mean fluorescent intensity of 3E11 scFv binding in HepG2 and HepG2-sh57 cells were calculated in C. D. scFv binding to surface of HepG2 (black line) and HepG2-sh57 (grey area) cells demonstrated by FACS. Cells were incubated with the indicated scFvs and detected by APC-conjugated anti-V5 mAb. E. Differential confocal immunofluorescence staining of scFv with HepG2 (GFP-negative) and HepG2.sh57 (GFP-positive) cells. The cellular mixture of HepG2 and HepG2.sh57 (1:1) were cultured in slice chamber. Cells were stained with the indicated scFv and detected by anti-V5 mAb followed by anti-mouse Alexa Fluor 546 secondary antibody.