WAVE complex rings observed for multiple cell lines, labeled subunits, microscopy techniques, and actin inhibitors. (A) Zoomed-in TIRF-SIM images of Hem1-EGFP rings across different drug conditions: 250 nM, 500 nM, 5 µM, and 10 µM (final) of latrunculin B (latB), latrunculin A (latA), and cytochalasin B (CB). Scale bar: 1 µm. (B) Boxplots (interquartile range) of ring diameters across drug conditions as measured in Fig. 2 D. Each condition has n = 10 cells except for latB 500 nM, which has n = 13 cells, and all cells are from at least three independent experiments per condition; ensemble histogram in Fig. 2 D. (C) Multiple super-resolution techniques in live or fixed cell conditions of Hem1 tagged with different fluorescent proteins all show ring structures. Microscopes and techniques used Airyscan 800 (Zeiss), Airyscan 880 (Zeiss), N-SIM (Nikon), SP8 STimulated Emission Depletion (STED; Leica), and photoactivated localization microscopy (PALM; B. Huang laboratory [UCSF] microscope). All cells treated with latrunculin B (500 nM). Scale bar: 1 µm. (D) Hem1 tagged with different fluorescent proteins (FP) show ring structures. All imaged with TIRF-SIM and treated with latrunculin B (500 nM). Scale bar: 1 µm. (E) Different EGFP-tagged WAVE complex subunits in B16F10 (Mus musculus skin melanoma cells), HUVECs, and U2OS (Homo sapiens bone osteosarcoma) cell lines show ring structures. All cells treated with latrunculin B (500 nM). TIRF-SIM imaging; scale bar: 1 µm.