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Fig. S1 Live cell imaging. For live cell imaging, the SK-BR-3 cells were cultured in 18 mm coverslips and washed twice with phenol red free RPMI (Sigma) containing 1 % glucose, 25 mM HEPES and 1 % BSA supplemented with 100 U/ml pencillin and 100 μg/ml streptomycin. The EC-Fc BNC tagged with RITC was incubated on ice for 30 min prior to the visualization in order to enable the binding of the multivalent forms of BNC to the cell surface. The cells were then washed thrice with warm media and were visualized in 100X 1.3 N.A. oil immersion objectiveunder Olympus IX81 inverted microscope (Olympus) with temperature control followed by data acquisition with Metamorph software (Molecular Devices). The imaging was carried out with DP71 cooled CCD camera attached to the microscope and the fluorescence of RITC was acquired along with the DIC field image. The time-lapse imaging was carried out for one hour with time intervals of 5 minutes each. In Metamorph software, each channel was split and overlayed separately. The acquired stacks were analyzed with MBF Image J.

Fig. S2 Microarray analysis. Expression of genes in SK-BR-3 cells was analyzed by microarray procedure. Total RNA preparation and analysis was performed as described previously [46, 47]. DNA microarray was carrying 1,795-oligonucleotide probes for human cell surface proteins. cDNAs were synthesized with Superescript II reverse transcriptase (Invitrogen) with oligo dT primers. Amino-allyl-dUTP was incorporated into cDNAs followed by coupling with Cy-3 dye (Ambion, TX, USA) and were processed for hybridization at 55°C for 15 hours. The fluorescent images for the hybridization were captured using FLA8000 scanner (Fuji Film, Japan) and analyzed with GenePix Pro5.1 software (Axon Instruments, CA). We found caveolins, Cav-1 and 2, and claudin 16 (CLDN-16) were downregulated in SK-BR-3 cells when compared to SK-OV-3 cells while ErbB2 and GAPDH expression were equally expressed in both the cell lines. RT-PCR (Fig. S1A) and qRT-PCR (Fig. S1B) further confirmed these results. The conditions for the RT-PCR are as follows: 94°C for 5min; followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec and 72°C for 7 minutes. Quantitative real time PCR was performed with SYBR Green Realtime Master Mix (Toyobo) in triplicates containing 5ng of cDNA along with 400 nM primers using LightCycler™ (Roche). The thermal cycling condition was as follows: 95°C for 1 min followed by 40 cycles of 95°C -10 sec, 55°C for 10 sec, 72°C for 25 sec and 60°C for 1 min. The following sets of the primers were used for the PCR reaction. Claudin-16, (Forward) 5′- GCTTGCCACAATGAGGGATCT-3′, (Reverse) 5′-TGACTTGGCCATGGAAACACC-3′; Cav-1 (Forward) 5′-GACTCGGAGGGACATCTCTAC-3′, (Reverse) 5′-GTTGATGCGGACATTGCTGA-3′; Cav-2, (Forward) 5′-ACGTACAGCTCTTCATGGAC-3′ (Reverse) 5′-CAGTTGCAGGCTGACAGAAG-3′.

Fig. S3 Time dependent colocalization of EC-Fc BNC in early endosomes. Colocalization of EC-Fc/BNC-RITC with EEA-1 was found in the early endosomes. SK-BR-3 cells were treated with EC-Fc/BNC-RITC for 5, 30, 90 min, and the colocalization of the EC-Fc/BNC-RITC with the early endosomal marker, EEA-1 was assessed with anti EEA-1 antibody (BD Biosciences) and the secondary antibody labeled with AlexaFlour-488 (Molecular Probes). Error bars indicate standard error. JACoP plugin in Image J was employed for percentage colocalization assessment. Percentage colocalization was found to be maximum up to approximately 70% by 30 min. The data represented here is the representative of two independent experiments.

Fig. S4 Binding of EC-Fc BNC to the cell surface after treatment with varying concentration of mβCD. The binding of EC-Fc/BNC’s to ErbB2, in the presence of 2 to 10 mM of cholesterol dislodging oligosaccharide-mβCD, was not affected. SK-BR-3 cells were treated with 5 and 10 mM of mβCD for 30 minutes followed by treatment with EC-Fc/BNC labeled with RITC are shown in Figure S2. The cells were stained with anti ErbB2 antibody (green) and red spots indicate EC-Fc/BNC labeled with RITC. Bars depict 10 μm.

Fig. S5 FACS analysis for the surface binding and internalization of EC-Fc BNC with inhibitor treatment. Surface binding and internalization of EC-Fc/BNC labeled with FITC in SK-BR-3 cells was quantified using FACS analysis. First, the binding of EC-Fc/BNC on SK-BR-3 cells was treated with or without 5 mM of mβCD on ice for 30 min. The cells were then fixed and analyzed with FACS to determine the surface bound fraction (Fig. S5A). The histograms showed overlapping of the peak tops indicating that the surface binding is majorly unaffected by the mβCD treatment. The peak (blue), which corresponds to approximately 20% of the total cell counts, orienting towards the untreated cells (grey peak) indicates the presence of a different population of cells that are incapable of binding to EC-Fc/BNC after mβCD treatment. Simultaneously, another set cells were treated with EC-Fc/BNC labeled with FITC in the presence or absence of 5 mM of mβCD at 37°C for 30 minutes. Cells were then trypsinized to remove the surface fraction, fixed and permeabilized, and the internalized fraction was analyzed adopting the same procedure with FACS (Fig. S5B, C). mβCD treated SK-BR-3 cells showed a peak point in the histogram which overlaid the peak area of untreated cells indicating the mβCD inhibits the internalization, while the cells without mβCD treatment drifted from the untreated cells towards the right. Total intensity of the internalized fraction was assessed from the FACS data were plotted on to a graph (Fig. S5). mβCD suppressed the internalization to approximately 50% when compared to the absence of mβCD treatment. M: mβCD; T: trypsin. Error bars indicate standard error.

FilenameFormatSizeDescription
JCMM_1277_sm_Movie.avi29795KSupporting info item
JCMM_1277_sm_SuppMat.doc38KSupporting info item

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