Dyngo 4a treatment affects cell morphology and F-actin. (A) Example of basal PM retraction after Dyngo 4a addition. White area depicts the area retracted between time point 0 to 10 min. Red area depicts the area retracted between time point 10 to 30 min. Yellow outline illustrates the basal PM surface area after 30 min treatment with Dyngo 4a. Scale bar, 10 μm. (B) Representative confocal image of basal membrane in Dyn2-GFP-Cav1-mCh cells treated with DMSO (Ctrl) or Dyngo 4a for 30 min and incubated with the F-actin marker SiR-actin. Scale bar, 20 μm. (C) Quantification of percentage of caveolae positive for Dyn2-GFP after 30 min Dyngo 4a treatment. Data are shown as scatter dot plot, mean ± SEM. (D) Representative immunofluorescent staining of Dyn2-GFP-Cav1-mCh cells. Cells transiently expressing EHD2-BFP were treated with DMSO (Ctrl) or Dyngo 4a for 30 min and then fixed and stained with cavin1 antibody (magenta). Yellow arrows depict colocalizing structures and green arrow depicts only Dyn2-GFP–positive structure. Scale bar, 10 μm. (E) Representative image of fixed Dyn2-GFP-Cav1-mCh cells treated with DMSO (Ctrl), Dyngo 4a, or Ryngo before 10 min incubation with Alexa fluor 647 conjugated Tf (5 μg/ml) at 37°C. Scale bar, 10 μm. Quantification show relative Tf-647 fluorescent intensity/microns2. Significance was assessed using t test, ***P ≤ 0.001.
