Figure S4.
Supplemental IRSp53 data. (A) dHL60 cell with high expression of IRSp53-EGFP stimulated with chemoattractant (10 nM fMLP) shows that high levels of IRSp53 induce ring structures even in the presence of an intact actin cytoskeleton. TIRF-SIM imaging; scale bars: 5 µm (left) and 1 µm (inset). See Video 6. (B) Graph comparing the diameters of the WAVE complex rings with F-actin inhibition to IRSp53 rings with (+) or without (−) F-actin inhibition (500 nM latrunculin B). Diameters measured in the same fashion shown in Fig. 2 D. Violin plot of rings; IRSp53 without (−) F-actin inhibition n = 157 from 10 cells, IRSp53 with (+) F-actin inhibition n = 483 from 10 cells, the WAVE complex (+) F-actin inhibition n = 202 from 13 cells; cells pooled from at least three independent experiments per condition; Kruskal–Wallis test; nonsignificant P = 0.56 > 0.05. (C) IRSp53-EGFP structure/function expression levels are comparable across cell lines. Left: IRSp53 structure/function constructs in wild type cells. Right: Full-length IRSp53 in wild type and WAVE complex-null HL60s. Flow cytometry data graphed with FlowJo. (D) Images of HUVECs expressing full-length IRSp53 (left) or I-BAR–only domain (right) at Y27632-induced TEMs. Spinning-disk confocal imaging; red arrows point to a TEM; scale bar: 5 µm. See Video 4. Graph: Violin plot of the enrichment of full-length IRSp53 and I-BAR domain at TEMs; measurements made in the same fashion as Fig. 5 B. Each time point across TEM closure was considered as a single data point: full-length n = 186 and I-BAR n = 291, both from four TEMs from at least three independent experiments per condition; Mann–Whitney test of significance; two-tailed; ****, P < 0.0001. (E) Characterization of CRISPR/Cas9-mediated IRSp53 knockout in HL60s. Left: Immunoblot of IRSp53 and β-actin (loading control) in wild type and IRSp53-null clonal HL60 line. Right: TOPO alignment of the gene-edited region of exon 2 reveals indels of the IRSp53-null clonal HL60 line. neg., negative.

Supplemental IRSp53 data. (A) dHL60 cell with high expression of IRSp53-EGFP stimulated with chemoattractant (10 nM fMLP) shows that high levels of IRSp53 induce ring structures even in the presence of an intact actin cytoskeleton. TIRF-SIM imaging; scale bars: 5 µm (left) and 1 µm (inset). See Video 6. (B) Graph comparing the diameters of the WAVE complex rings with F-actin inhibition to IRSp53 rings with (+) or without (−) F-actin inhibition (500 nM latrunculin B). Diameters measured in the same fashion shown in Fig. 2 D. Violin plot of rings; IRSp53 without (−) F-actin inhibition n = 157 from 10 cells, IRSp53 with (+) F-actin inhibition n = 483 from 10 cells, the WAVE complex (+) F-actin inhibition n = 202 from 13 cells; cells pooled from at least three independent experiments per condition; Kruskal–Wallis test; nonsignificant P = 0.56 > 0.05. (C) IRSp53-EGFP structure/function expression levels are comparable across cell lines. Left: IRSp53 structure/function constructs in wild type cells. Right: Full-length IRSp53 in wild type and WAVE complex-null HL60s. Flow cytometry data graphed with FlowJo. (D) Images of HUVECs expressing full-length IRSp53 (left) or I-BAR–only domain (right) at Y27632-induced TEMs. Spinning-disk confocal imaging; red arrows point to a TEM; scale bar: 5 µm. See Video 4. Graph: Violin plot of the enrichment of full-length IRSp53 and I-BAR domain at TEMs; measurements made in the same fashion as Fig. 5 B. Each time point across TEM closure was considered as a single data point: full-length n = 186 and I-BAR n = 291, both from four TEMs from at least three independent experiments per condition; Mann–Whitney test of significance; two-tailed; ****, P < 0.0001. (E) Characterization of CRISPR/Cas9-mediated IRSp53 knockout in HL60s. Left: Immunoblot of IRSp53 and β-actin (loading control) in wild type and IRSp53-null clonal HL60 line. Right: TOPO alignment of the gene-edited region of exon 2 reveals indels of the IRSp53-null clonal HL60 line. neg., negative.

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