Fig. 2.

HA-binding heterogeneity arises from distinct abilities of HA receptor-positive subpopulations to interact with HA. The fluorescent characteristics of cells after incubation with CD44 and RHAMM antibodies (Ab) in the presence or absence of F-HA are shown as dot plots; (A, i) Background fluorescent level of control cells without Ab. (A, ii) CD44 Ab. (A, iii) RHAMM Ab. (B) Demonstration of F-HA binding to live cells coexpressing CD44 and RHAMM. (B, i) F-HA–binding pattern. (B, ii and iii) Multiplexed detection of F-HA interaction with RHAMM and CD44. n = 3. (C, i) Fluorescence images of MDA-MB-231 cells cultured in 2D and then exposed to F-HA in the absence of a RHAMM mimetic peptide, scrambled peptide, or no peptide (Upper), and quantification of F-HA signal in the nucleus (Lower). n = 12–63. (Scale bars: 150 µm in C, i, Upper and 50 µm in C, ii.) Error bars represent SE. (C, ii) Heat maps of F-HA uptake by MDA-MB-231 cells in the presence of antibodies to isotype matched nonimmune IgG or to CD44. (D) Fluorescent images of F-HA uptake in 3D (i), 2D (ii), and subcellular localization (Insets). n = 5. (Scale bars: 100 µm in D, i and 50 µm in D, ii.) Images in D, ii were taken from different regions of the same uptake assay and merged side by side. (E) TEM analysis of accumulated Au-HA nanoparticles (shown by arrows) in the nucleus. (F) FACS analysis of F-HA binding at 4 °C vs. 37 °C. n = 3.