Figure 1.

Binding of HA to LYVE-1 in HDLECs is increased upon actin depolymerization. A, confocal z-stack projection of hLYVE-1–transfected HDLECs, stained for LYVE-1 using LYVE-1 mAb/Alexa Fluor 594 conjugate (magenta) and for actin using Abberior STAR 635 phalloidin (cyan) (scale bar, 20 μm). B, super-resolution STED imaging of a 10 × 10-μm region of the HDLEC cell surface (yellow boxed area from A) to assess the localization of LYVE-1 (magenta) and actin (cyan). LYVE-1 was mostly located within corrals enclosed by the cortical actin meshwork and in a small number of cases appeared to overlie actin filaments themselves (yellow arrowheads) with a Pearson's correlation value of 0.1 ± 0.07. The scale bar (2 μm) applies to all three images shown. C, confocal z-stack projections showing binding of fluorescent bHA-coated beads (yellow) to monolayers of either DMSO (control)–, Cyto D–, Lat B–, or CK-666–treated HDLECs (magenta) either alone (top) or in the presence of a LYVE-1 HA blocking antibody (bottom). Images are representative of three experimental replicates. The scale bar (50 μm) applies to all eight images shown. Some of these images are also reproduced at higher magnification in Fig. S1 (see “Results”). DAPI, 4′,6-diamidino-2-phenylindole. D, quantification of bHA-coated bead binding from the data in C performed in the presence or absence of LYVE-1 HA-blocking antibody (top and bottom panels, respectively), indicating the sum from experimental replicates (10 fields/condition, n = 3 and n = 4 for CK-666 experimental replicated) with S.E. (error bars) and significance indicated by unpaired t test (Cyto D = 0.0129, Lat B = 0.0335, CK-666 = 0.1103; with blocking DMSO = 0.0007, Cyto D = 0.0229, Lat B = <0.0001, CK-666 = 0.0122). Blocking data are normalized to their respective nonblocking data. E, FACS histograms showing quantification of bHA:SA488 complex binding (orange and green) to HDLECs treated with either Cyto D, Lat B, or CK-666 or to untreated HDLEC controls (blue). Insets, median fluorescent intensity plots, normalized to untreated controls. Data are the mean ± S.E. from three replicate experiments. Statistics are compared using one-way analysis of variance with Dunnett's multiple comparison; p = 0.0211 for Cyto D, p = 0.0080 for Lat B, p = 0.0015 for CK-666. F, corresponding controls for the experiments in E performed in the presence or absence of excess unlabeled high-molecular-weight HA (xs HMWHA). Insets, median intensity plots normalized to the untreated conditions. Data are from two replicates. *,p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant.