Figure 2
Trastuzumab incubation reduces membrane-bound HER2 levels, and changes the cell membrane topography by abolishing membrane ruffles. Shown are images of the QD-fluorescence channel measuring membrane-bound HER2 represented in false color. (A) SKBR3 cells were first incubated for 10 min at 37 °C to label all membrane-bound HER2 with the Affibody, followed by 5 min of incubation with 10 μg/mL of trastuzumab at 37 °C, fixation, and QD labeling. Compared to control cells (see Fig. 1A), the 5 min drug incubation led to a loss of the typically higher HER2 densities on the cell peripheries and on membrane ruffles, as reflected by the absence of membrane areas with orange and yellow color. (B) Results of a pulse-chase experiment in which the Affibody-labeled cells were first incubated with trastuzumab as in A, but for 20 min, followed by wash steps and a 2 h chase period in medium at 37 °C, prior to fixation and QD labeling. Compared to A, the level of membrane-bound HER2 has reduced, membrane ruffles have completely disappeared, and a fraction of the cells have reacted by marked constriction. (C) Extension of the drug incubation to 1 h (without a subsequent chase time) leads to further reduction of membrane HER2 levels. (D) The depicted SKBR3 cells were first incubated for 60 min with trastuzumab, and then labeled with Affibody. This led to similar changes as shown in (C), thus, no reversal of the drug effect by reappearing/recycled HER2 was found. The images were recorded and represented with the same settings. Scale bars: 100 μm.
