1606 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 6. Cell-penetrating Her218-selective 4MH2 knocks-down endogenous Her2 expression in Her2-overexpressing SK-BR-3 cells. SK-BR-3 cells were untreated (‘control’) or treated with 3D8 VLs (10 mM) for 2 h at 37 C, or transfected with Her218-siRNA (500 nM). Her2 expression was monitored at the cell-surface by ﬂow cytometry (A), at the mRNA level by RT-PCR (B) and at the protein level by western blotting (C) at indicated periods of post-treatment with 3D8 VLs or post-transfection with Her218-siRNA (500 nM). Endogenous b-actin served as the protein loading and mRNA abundance control for western blotting and RT-PCR analyses, respectively. ‘Control’ indicates untreated cells. Figure 7. Cell-penetrating Her218-selective 4MH2 induces apoptotic cell death of Her2-overexpressing cells. (A) Cell viability assay of Her2-overexpressing SK-BR-3 and MDA-MB-231 cells and Her2-negative HeLa cells at the indicated periods of post-treatment with 3D8 VLs (10 mM) for 2 h at 37 C or post-transfection of Her218-siRNA (500 nM), as monitored by the MTT assay. Percent cell viability was calculated with respect to the untreated control cells. (B) Flow cytometry analysis of Annexin-V-FITC and PI staining of Her2-overexpressing SK-BR-3 and MDA-MB-231 cells at 12 h post-treatment with medium only (‘control’) or with 3D8 VLs (10 mM) for 2 h at 37 C, or at 12 h post-transfection with Her218-siRNA (500 nM). In the dot plots, Annexin-V-FITC+/PI cells (lower right quadrant, % shown) and Annexin-V-FITC+/PI+ cells (upper right quadrant, % shown) are considered as ‘early apoptotic’ and ‘dead’ cells, respectively. cell death exerted by 4MH2 ( 75%) and other 3D8 VLs ( 30–40%) in Her2-overexpressing cells could be attributed the apoptotic cell death induced by the Her2 gene-silencing eﬀects of 4MH2. DISCUSSION Antibodies that normally lack the ability to penetrate into living cells have been therapeutically used as targeting proteins, including mainly soluble or cell-surface