Nucleic Acids Research, 2010, Vol. 38, No. 5 1605 Figure 5. Target gene silencing activity of cell-penetrating 3D8 VL variants in HeLa cells expressing exogenous targeted genes. (A–C) HeLa cells were untransfected (‘control’) or transfected with plasmids encoding EGFP or G18-EGFP, and 12 h later either untreated or treated at 37 C for 2 h with 3D8 VL WT (10 mM) and G18-selective 4MG3 (10 mM) and 4MG5 (10 mM), and further incubated for 12 h before EGFP expression analyses by ﬂow cytometry (A), RT–PCR (B) and western blotting (C). (D and E) Her2-negative HeLa cells were untransfected (‘control’) or transfected with a plasmid encoding the full-length Her2 gene, and 24 h later were either untreated or treated at 37 C for 2 h with 3D8 VL WT (10 mM) and Her218- selective 4MH2 (10 mM). After further incubation for 24 or 48 h, Her2 expression was analyzed by RT-PCR (D) and western blotting (E). After 24 h post-transfection with Her2 gene, HeLa cells were also transfected with Her218-siRNA (500 nM) prior to analyses of Her2 expression at the indicated time. (B–E) Endogenous b-actin served as the protein loading and mRNA abundance control for western blotting and RT-PCR analyses, respectively. Like the 3D8 VL variants internalized as above, the co-expression of 3D8 VL variants with target sequence- carrying genes in HeLa cells selectively down-regulated the expression levels of targeted proteins by reducing their mRNA levels without signiﬁcantly aﬀecting oﬀ- target genes (Supplementary Figure S6). This result demonstrated that cytosolically expressed 3D8 VL variants also selectively degrade their respective target mRNAs to downregulate the expression of target proteins without signiﬁcant eﬀects on oﬀ-targets. Her218-selective 4MH2 eﬀectively knocks-down endogenous Her2 expression to induce apoptotic cell death in Her2-overexpressing cells To determine whether Her218-selective 4MH2 could knockdown the endogenous Her2 transcript by cellular internalization, Her2-overexpressing breast carcinoma SK-BR-3 cells (22) were treated with 4MH2, including other 3D8 VLs for comparisons. 3D8 VL WT and G18- selective 4MG3 and 4MG5 exhibited only slight decreases in Her2 expression after 24 h post-treatment (Figure 6A– C), which was most likely due to non-speciﬁc oﬀ-target eﬀects. In contrast, 4MH2 greatly reduced the cell surface expression and mRNA levels of Her2 starting 2 h post-treatment, completely abolishing Her2 mRNA detec- tion by 24 h, with only a slight reduction in the mRNA level of oﬀ-target b-actin at 48 h (Figure 6A–C). Compared with 4MH2, Her218-siRNA-mediated gene silencing of Her2 was slower and less eﬃcient, decreasing the cell surface expression and mRNA levels after 24 h post-transfection. While elevated Her2 expression correlates with malig- nant potential and poor prognosis of several types of car- cinomas, silencing of Her2 by siRNA (22,23) or its neutralization by intracellular antibody (intrabody) (27) resulted in growth inhibition and cell death in Her2- overexpressing tumor cells, including SK-BR-3 cells. To investigate whether 4MH2-mediated Her2 gene silencing can exert the similar activity, we treated Her2- overexpressing SK-BR-3 and MDA-MB-231 cells with Her218-selective 4MH2 and then monitored for the cell death, including Her2-negative HeLa cells (27) as a control. 4MH2 treatment signiﬁcantly induced cell death which was proportional to the post-treatment period in SK-BR-3 ( 75% after 48 h) and MDA-MB-231 ( 65% after 72 h), but which was moderate in HeLa cells ( 30% after 72 h) (Figure 7A). Transfected Her218-siRNA also induced cell death in Her2-overexpressing cells without signiﬁcant cytotoxicity to HeLa cells. However, Her18- siRNA-mediated cell death of Her2-overexpressing cells was less eﬃcient than 4MH2 during the same period, which is consistent with the relative knockdown eﬃciency of Her2 (Figure 6). To elucidate whether apoptosis is the principle mecha- nism of 4MH2-mediated cell death, cells exposed to 4MH2 were labeled by dual staining of Annexin-V-FITC and propidium iodide (PI) and then analyzed by ﬂow cytometry (19). SK-BR-3 and MDA-MB-231 cells treated with 4MH2 were signiﬁcantly labeled as early apoptotic cells by Annexin-V-FITC positive staining (Figure 7B), similar to Her218-siRNA, suggesting that 4MH2 induced apoptotic cell death in the Her2- overexpressing cells. 3D8 VL WT and G18-selective 4MG3 and 4MG5 caused 30–40% cell death after 72 h post-treatment, regardless of the Her2 cellular expression level, demonstrating that 3D8 VLs caused some cytotoxicity, which was probably due to non-speciﬁc cellular RNA hydrolysis (7). However, diﬀerences in the