1598 Nucleic Acids Research, 2010, Vol. 38, No. 5 ﬂuorescence arbitrary value, 0. Arbitrary ﬂuorescence value was converted into substrate concentration using the standard curve, in which ﬂuorescence intensity changes were plotted as a function of substrate concentra- tions that were completely digested by DNase I for ss-DNA substrates and bovine pancreatic RNase A (Invitrogen) for ss-RNA substrates (18). Then initial rate constants (V) at each substrate concentration were deter- mined by linear regression over the initial linear regions (between 0 and 500 s) of the normalized data using Softmax pro software (Molecular Devices). The apparent enzymatic kinetic parameters, Km and Vmax, were determined by ﬁtting the initial rate constants (V) versus substrate concentrations (S) into the Michaelis– Menten equation [V = (Vmax[S])/(Km + [S])] and Lineweaver–Burk equation using Sigmaplot 2002 software (SPSS Inc.) (12,18). All experiments were per- formed in triplicate and the results are expressed as mean ± standard deviation (SD). Signiﬁcant diﬀerence was determined using two-tailed Student’s t-test versus the control N18 substrate on Excel software (Microsoft Inc.). A P-value of < 0.01 was taken as statistically signiﬁcant. Cell penetration Human cervical carcinoma HeLa, breast carcinoma SK- BR-3 and MDA-MB-231 cells were from American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (Dulbecco’s Modiﬁed Eagle Medium) supple- mented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) in a humidiﬁed 5% CO2/95% air atmosphere at 37 C (7,15,19). All of the cell lines were routinely screened for Mycoplasma contamination. Cell penetration experiments and subsequent various analyses were performed as described previously (7,12,15). Cells were seeded at a density of 5 105 cells/well in 6-well plate (for ﬂow cytometry, RT–PCR, western blotting, and cell viability assays) or 5 104 cells/well in 24-well plate over glass coverslips (for confocal microscopy) the day before use, and pre-incubated in serum-free DMEM for 30 min at 37 C prior to the treatment of proteins and/or reagents. Then the cells were incubated with 3D8 VLs (each 10 mM) for 2 h at 37 C with or without pre-treatment with 100 IU/ ml soluble heparin, chloropromazine (10 mg/ml), methyl- b-cyclodextrin (5 mM) and cytochalasin D (1 mg/ml) for 30 min at 37 C, washed and then further incubated for the indicated periods prior to subjecting to vari- ous analyses, as speciﬁed in ﬁgure legends. Cell viability was analyzed using a colorimetric MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)- based cell growth determination kit (Sigma) (7,15,19). The percentage of apoptotic cells was quantiﬁed by ﬂow cytometry after ﬂuorescent staining with Annexin-V-FITC and propidium iodide (PI) (19). Cell transfection All transfections were performed using Lipofectamine 2000 (Invitrogen Inc.) with plasmid DNA (each 0.5 mg) or Her218-siRNA (500 nM) following the manufacturer’s instructions (7). For HeLa cells transfected with plasmid of pEGFP-N1, pG18-EGFP, or pcDNA3-Her2, the cells were treated for 2 h at 37 C with 3D8 VLs (10 mM) after 12 or 24 h post-transfection, washed and then further incubated for the indicated periods prior to subjecting to various analyses. In experiment with SK-BR-3 cells, the cells grown 50% conﬂuency ( 5 105 cells in 6-well plates) were transfected with Her218-siRNA as above, prior to subjecting to various analyses. Confocal ﬂuorescence microscopy Confocal ﬂuorescence microscopic analyses of the cells were performed as described previously (7,15). Brieﬂy, the cells were washed twice with cold PBS, ﬁxed with 2% paraformaldehyde in PBS for 10 min at 25 C, and then permeabilized with Perm-buﬀer (1% BSA, 0.1% saponine, 0.1% sodium azide in PBS) for 10 min at 25 C. For staining of internalized 3D8 VLs, the cells were blocked with 2% BSA in PBS for 1 h, incubated with rabbit anti-3D8 polyclonal antibodies (7), followed by TRITC-anti-rabbit IgG. Nucleus was stained with DAPI (40,6-diamidino-2-phenylindole dihydrochloride) (Vector Labs) during the last 10 min of incubation at 25 C. Cells on coverslip were mounted in Vectashield anti-fade mounting medium (Vector Labs), and observed with Zeiss LSM 510 laser confocal microscope and analyzed with Carl Zeiss LSM Immage software. Flow cytometry For the quantiﬁcation of EGFP expression in HeLa cells treated as above, the suspended cells with trypsin were washed twice with 1 ml ice-cold PBS and then directly subjected to ﬂow cytometry on a Becton Dickinson FACSCaliburTM (7,15). For the cells transfected with EGFP and/or treated with 3D8 VLs (10 mM) for cellular internalization as described above, the suspended cells with trypsin were treated once more with 0.1% trypsin for 3 min at 37 C to wash oﬀ the surface bound proteins (7). After washings with ice-cold PBS once, the cells were ﬁxed and permeabilized as the procedures described in above ‘confocal ﬂuorescence microscopy’. The cells were washed with ice-cold PBS twice, labeled with rabbit anti- 3D8 polyclonal antibodies followed by TRITC-anti- rabbit, and then analyzed using the ﬂow cytometry. In experiment with the SK-BR-3 cells transfected with Her218-siRNA or exposed to 3D8 VLs as above, cell- surface expression levels of Her2 were monitored by immunoﬂuorescent labeling of rabbit anti-Her2 mAb (1 h on ice) and then goat FITC-labeled anti-rabbit IgG (1 h on ice) (7,15). For each test, 1 104 cells were analyzed. Reverse transcription (RT) PCR To monitor mRNA levels in cells, semi-quantitative RT– PCR was performed following the standard procedures (7,20). Total cellular RNA was extracted from speciﬁed cells, using the Trizol (Gibco Invitrogen), according to the manufacture’s instruction. Aliquots ( 1 mg) of total RNA were used for ﬁrst strand complimentary DNA (cDNA) synthesis using PreMix cDNA synthesis kit