1604 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 4. 3D8 VL variants penetrate into living cells by interaction with heparin sulfate proteoglycan on the cell surface and localize dominantly in the cytosol. (A) Cellular internalization of 3D8 VLs in HeLa (left panel) and SK-BR-3 (right panel) cells, monitored by ﬂow cytometry. HeLa and SK-BR-3 cells were treated with each 3D8 VL (10 mM) for 2 h at 37 C and then with trypsin to remove surface bound proteins before immunoﬂuorescent labeling of 3D8 VLs with rabbit anti-3D8 polyclonal antibodies and TRITC-labeled anti-rabbit IgG for ﬂow cytometry. (B) Internalization and subcellular localization of 3D8 VLs in HeLa cells, untreated (‘control’) or treated with 10 mM 3D8 VLs at 37 C for 2 h prior to analysis by confocal ﬂuorescence microscopy. 3D8 VLs were stained with rabbit anti-3D8 polyclonal antibodies and TRITC-labeled anti-rabbit IgG (Red). Blue color depicts DAPI-stained nuclei. Centered single confocal sections are shown. Magniﬁcation, 400. (C) Eﬀect of pre-treatment of soluble heparin or speciﬁc endocytosis inhibitors on the 3D8 VLs cellular uptake, analyzed by ﬂow cytometry. HeLa cells were incubated with 3D8 VLs (10 mM) for 2 h at 37 C with or without pre-treatment of soluble heparin (100 IU/ml), CPZ (10 mg/ml), MbCD (5 mM) and Cyt-D (1 mg/ml) for 30 min prior to ﬂow cytometric analyses. a signiﬁcant reduction in the amount of 3D8 VLs internalized (Figure 4C). These results strongly indicated that 3D8 VLs are internalized via caveolae/lipid raft- mediated endocytosis similar to 3D8 scFv (7). Cell penetrating 3D8 VL variants selectively knockdown target genes The penetration of 3D8 VLs into living cells and their dominant accumulation in the cytosol prompted us to examine whether 3D8 VLs could selectively degrade mRNAs carrying targeted sequences. When evaluated by EGFP reporter gene with N-terminal G18 (G18-EGFP) in HeLa cells, G18-selective 4MG3 and 4MG5 exhibited sig- niﬁcant reductions in ﬂuorescence intensity for G18-EGFP but not for intact EGFP (Figure 5A). Semi-quantitative RT-PCRs (20) demonstrated that 4MG3 and 4MG5 selectively decreased only the amount of G18-EGFP mRNA, without signiﬁcant eﬀects on mRNAs encoding intact EGFP or a house-keeping gene, b-actin (Figure 5B). This result was also reﬂected at the protein level (Figure 5C). In contrast, 3D8 VL WT did not aﬀect the expression levels of intact EGFP or G18-EGFP. Her218-selective 4MH2 was incubated with HeLa cells transfected with the full-length Her2 gene. For compari- son, a siRNA targeting the same sequence of Her218 in the Her2 gene (Her218-siRNA) was transfected into Her2- transfected HeLa cells. Both 4MH2 and Her218-siRNA, but not 3D8 VL WT, selectively down-regulated Her2 expression at the mRNA and protein levels, without aﬀecting endogenous b-actin expression (Figure 5D and E). Her2 gene silencing by 4MH2 at the mRNA and protein levels was more eﬀective at the earlier time-point of 24 h post-treatment than Her218-siRNA, which exerted substantial Her2 down-regulation after 48 h post- transfection.