Dynamic AFM imaging of effects of HCQ on AnxA5 crystallization.(A) The binding of β₂GPI to phospholipid bilayer after overnight incubation at 4°C resulted in formation of clusters of the protein. An arrow indicates one of these clusters as a representative reference point for the subsequent sequences. (B) The binding of aPL mAb, IS4, to β₂GPI clusters showing the formation of aPL IgG-β₂GPI complexes with increased heights and thickening of each of the clusters (arrow). (C) Addition of HCQ and AnxA5 resulted in the erosion of the complexes (arrow), as previously described, and (D) formation of “cobblestones” of Anx A5 that formed over and around the remnants of the aPL mAb-β₂GPI complexes (arrow). The appearances of the AnxA5 patches are identical to the formations shown in the endpoint experiment of Figure 5B. All above 15 μm × 15-μm amplitude images were electronically zoomed from an original 30 μm × 30-μm scan.