Panel a shows Western blots with labels for different proteins and shRNA treatments, indicating knockdown efficiency. Panel b displays widefield images of wound migration at two time points, with Hoechst staining. Panel c presents images of collectively migrating cells stained for nuclei, junctions, and Golgi. Panel d and e feature dot plots showing the percentage of AAJs positive for specific proteins, with axes labeled for percentage and protein types. Panel f shows images of migrating cells with different treatments, stained for nuclei, junctions, and Golgi. Panel g contains phase contrast images of wound migration at two time points, comparing control and treated cells.
Validation of shRNAs for BAR proteins, BAR-GFP overexpression, and DNM2 knockdowns. (a) Representative western blots showing shRNA knockdown efficiency for the indicated BAR proteins with targeting shRNAs. Asterisk indicates an shRNA whose knockdown is lethal to cells. (b) Representative widefield images of wound migration at t = 0 and t = 7 h after scratch in HUVECs depleted with shRNAs of indicated BAR proteins stained with Hoechst. See also Video 4. (c) Representative images of collectively migrating HUVECs transduced with shRNAs targeting the indicated BAR proteins, fixed 6 h after scratch wound formation. Nuclei are stained with DAPI (blue), junctions with VE-cadherin (magenta), and Golgi with GM130 (green). (d and e) Dot plots showing the percentage of AAJs per cell that are positive for BIN1-GFP or SNX9-GFP (d) and dot plots showing the percentage of AAJs per cell that are positive for PACSIN2 in cells expressing BIN-GFP or GFP-SNX9 (e). Data are presented as mean ± SD. Circles indicate individual data points and triangles indicate means of individual experiments with colors corresponding to the experimental replicates. Statistical analysis was performed using a two-sided Student’s t test. n = 29 cells for BIN1-GFP and 28 for GFP-SNX9 from four independent experiments. P values are indicated on the graph. (f) Representative images of migrating HUVECs transduced with shControl and shDNM2-1, fixed 6 h after scratch wound formation. Nuclei are stained with DAPI (blue), junctions with VE-cadherin (magenta), and Golgi with GM130 (green). (g) Representative phase contrast images of wound migration at t = 0 and t = 9 h after scratch in HUVECs transduced with shControl and shDNM2-1. See also Video 13. Scale bars = 100 and 50 μm. Source data are available for this figure: SourceData FS4.