Error bars denote standard deviation

Error bars denote standard deviation. (TIF) Click here for additional data file.(530K, tif) Figure S2 Comparison of free C225 antibody with C225-NP on their ability to induce apoptosis in lung tumor cells. cells in the subG1 phase compared to treatment with C225 antibody at all concentrations tested. *P-value 0.05 vs same concentrations of C225 antibody.(TIF) pone.0025507.s002.tif (62K) GUID:?BB708459-49E9-469E-B5DC-D7C20EB46ACD Figure S3: C225-NP induces autophagy in H1299 lung cancer cells. (a) Detection of GFP-LC3 dots in H1299 cells that were either not treated or treated with C225 antibody, IgG-NP or C225-NP (3109 particles) for 72 hrs on chamber slides. (b) Quantitative analysis showed C225-NP-treated HCC827 cells had higher number of GFP-LC3 dots in compared to all other treatment groups. Results shown are the means S.D. of three independent experiments. *P-value 0.05 vs untreated control, C225 antibody, and IgG-NP.(TIF) pone.0025507.s003.tif (178K) GUID:?F34C015F-BA64-4254-9904-A9CA3D605BCA Figure S4: Visualization, and determination of selective binding and uptake of C225-NP in H1299 cells. (a) In the right column cells were treated with C225 antibody (2 g/ml) for 15 min, and then incubated with either IgG-NP or C225-NP for additional 24 hrs. The left column shows cells which were not pre-treated with free antibodies. The slides were washed, fixed and imaged under dark-field microscopy. Binding and uptake of C225-NP was completely inhibited in the presence of C225 antibody. In IgG-NP-treated cells C225 antibody had no effect. Scale bar is 50 micron. (b) Inhibition effects of free C225 antibody on the cytotoxicity of C225-NP by pre-treatment with free C225 antibody. After treatment with C225 antibody (0.065 g/ml) for 6 hrs, the cells were treated with C225-NP for an additional 66 hrs. Cells treated for 66 hrs with C225-NP, C225 antibodies alone, non-conjugated NP (gold-iron: AuFe), and IgG-NP were used for comparison. Results shown are the means S.D. of three independent experiments. *P-value 0.05 vs AuFe alone, C225 antibody alone, IgG-NP or C225 antibody plus C225-NP. (c) Inhibition effects of pre-treatment with C225 antibody on C225-NP-induced apoptosis and autophagy. Cellular proteins were lysed after treatment with C225-NP (0.61010 particles) for 66 hrs in the presence or absence of free C225 antibody (0.065 g/ml). Proteins were separated by 7.5% or 15% SDS-PAGE, and immunoblotted with anti-PARP and anti-LC3 antibodies. The intensities of (??)-Huperzine A the amount of LC3-II bands were quantified by ImageJ software (National Institutes of Health). C225-NP-mediated activation of apoptosis and autophagy as indicated by cleavage of capase-3 and LC3-II respectively were markedly abrogated in the presence of free C225 antibody. PARP cleavage was not detectable in all of the groups.(TIF) pone.0025507.s004.tif (605K) GUID:?F6BF6C66-9AB6-493C-BB26-408AF0CF7CBF Figure S5: Nanoparticle size determination by transmission electron microscopy. Size analyses at lower and higher magnification showed antibody-conjugated NPs were 5411 nm in size. (c) Quantification of the number of cells with GFP-LC3 dots on untreated and treated NSCLC cells. The number of cells with GFP-LC3 dots was higher in C225-NP-treated HCC827 cells compared to all other treatment groups. In H520 cells DLL4 there was no increase in (??)-Huperzine A (??)-Huperzine A the number of GFP-LC3 dots when treated with C225-NP and compared to all other treatment groups. Results shown are the means S.D. of three independent experiments. *P-value 0.05 vs untreated control, C225 antibody, and IgG-NP on HCC827 cells. Recently, it has been shown that quantum dots induce size-dependent autophagy in human mesenchymal stem cells [19]. We therefore examined whether autophagy occurred in NSCLC cells after treatment with C225-NP. The green fluorescent protein (GFP)-tagged expression vector of LC3 is a useful tool for detecting autophagy [20]. On fluorescent microscopy, GFP-LC3-transfected HCC827 cells showed diffuse distribution of GFP-LC3 for untreated cells, and cells treated with Clone 225 antibody alone and with IgG-NP, whereas the cells treated with C225-NP showed GFP-LC3 punctate dots indicative of autophagic vacuoles ( Figure 4b ). The percentage of cells with GFP-LC3 punctate dots increased after treatment with C225-NP for HCC827 cells ( Figure 4c ; P 0.05). Induction of autophagy determined by GFP-LC-3 dots was also markedly increased in C225-NP-treated H1299 cells compared to other treatment groups (Figure S3). In contrast, the amount of GFP-LC3 punctate dots was not different in.