Figure S1.
WAVE complex rings are not due to overexpression of WAVE complex subunits and are devoid of F-actin.(A) HEM1 and WAVE2 expression levels in the Hem1-EGFP HL60 cell line used throughout the study. Left: Western blot of HEM1 and GAPDH in wild type, HEM1-null, and Hem1-EGFP HL60 cell lines. Right: Western blot of WAVE2 and GAPDH in wild type and Hem1-EGFP HL60 cell lines. (B) WAVE2 split-GFP endogenous knock-in sequences. In a GFP1-10–expressing cell line, CRISPR/Cas9 cuts the N terminus of WAVE2’s exon 2 and the cell repairs with a GFP11 donor flanked with WAVE2 homology arms. (C) Endogenous localization of WAVE2 in split-GFP knock-in dHL60 cells. Cells treated with chemoattractant (10 nM fMLP) or F-actin inhibitor (500 nM latrunculin B). TIRF-SIM imaging; scale bars: 5 µm and 1 µm (inset). Violin plot of the WAVE complex ring diameters in Hem1-EGFP and WAVE2 knock-in cells treated with F-actin inhibitor (500 nM latrunculin B); Hem1-EGFP n = 293 rings from 13 cells, WAVE2 knock-in n = 305 from 10 cells; cells pooled from at least two independent experiments per condition. Diameters measured in the same fashion shown in Fig. 2 D. (D) Images of dHL60 cells expressing Hem1-EGFP (green) treated with chemoattractant (100 nM fMLP; top) or F-actin inhibitor (5 µM latrunculin B; bottom) that were then fixed and stained with phalloidin (red). TIRF-SIM imaging; scale bars: 5 µm and 1 µm (insets). Convention resolution TIRF inset images are the sum-projection of the nine images (three phases × three angles) that construct TIRF-SIM images. Graph shows mean ± SEM of phalloidin intensity/area per cell; (−) F-actin inhibitor n = 14 cells, 5 µM latrunculin B F-actin inhibitor n = 10 cells, 500 nM latrunculin B F-actin inhibitor n = 10 cells; cells pooled from at least three independent experiments; multiple t tests comparing the phalloidin staining (signal) and a scrambled version of the phalloidin image (randomized); **, P = 0.003 < 0.01; ns, not significant; 5 µM F-actin inhibitor, P = 0.14 > 0.05; and 500 nM F-actin inhibitor, P = 0.17 > 0.05.

WAVE complex rings are not due to overexpression of WAVE complex subunits and are devoid of F-actin. (A) HEM1 and WAVE2 expression levels in the Hem1-EGFP HL60 cell line used throughout the study. Left: Western blot of HEM1 and GAPDH in wild type, HEM1-null, and Hem1-EGFP HL60 cell lines. Right: Western blot of WAVE2 and GAPDH in wild type and Hem1-EGFP HL60 cell lines. (B) WAVE2 split-GFP endogenous knock-in sequences. In a GFP1-10–expressing cell line, CRISPR/Cas9 cuts the N terminus of WAVE2’s exon 2 and the cell repairs with a GFP11 donor flanked with WAVE2 homology arms. (C) Endogenous localization of WAVE2 in split-GFP knock-in dHL60 cells. Cells treated with chemoattractant (10 nM fMLP) or F-actin inhibitor (500 nM latrunculin B). TIRF-SIM imaging; scale bars: 5 µm and 1 µm (inset). Violin plot of the WAVE complex ring diameters in Hem1-EGFP and WAVE2 knock-in cells treated with F-actin inhibitor (500 nM latrunculin B); Hem1-EGFP n = 293 rings from 13 cells, WAVE2 knock-in n = 305 from 10 cells; cells pooled from at least two independent experiments per condition. Diameters measured in the same fashion shown in Fig. 2 D. (D) Images of dHL60 cells expressing Hem1-EGFP (green) treated with chemoattractant (100 nM fMLP; top) or F-actin inhibitor (5 µM latrunculin B; bottom) that were then fixed and stained with phalloidin (red). TIRF-SIM imaging; scale bars: 5 µm and 1 µm (insets). Convention resolution TIRF inset images are the sum-projection of the nine images (three phases × three angles) that construct TIRF-SIM images. Graph shows mean ± SEM of phalloidin intensity/area per cell; (−) F-actin inhibitor n = 14 cells, 5 µM latrunculin B F-actin inhibitor n = 10 cells, 500 nM latrunculin B F-actin inhibitor n = 10 cells; cells pooled from at least three independent experiments; multiple t tests comparing the phalloidin staining (signal) and a scrambled version of the phalloidin image (randomized); **, P = 0.003 < 0.01; ns, not significant; 5 µM F-actin inhibitor, P = 0.14 > 0.05; and 500 nM F-actin inhibitor, P = 0.17 > 0.05.

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