Figure S4.
Early release of RUSH-α5 is adhesion dependent and polarized recruitment to protrusions is supported by endogenous integrin α5. (A–D) Validation of ITGA5 CRISPR-Cas9 KO U2OS cells. (A) Western blot analysis of WT and ITGA5 KO single cell clones showing the efficiency of the CRISPR-Cas9 ITGA5 KO in U2OS cells. (B) Genome sequence alignment of U2OS WT and ITGA5 KO clones with the ITGA5 WT sequence. The targeted exon and the gRNA used for CRISPR KO positions are indicated. (C) Representative flow cytometry analysis of cell surface integrin α5 in U2OS WT and ITGA5 KO clones. (D) Images of WT and ITGA5 KO U2OS clones stained for active integrin α5 (SNAKA51) and paxillin. Scale bar: 20 µm. (E) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in WT and ITGA5 KO U2OS cells transfected with RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test showed no significant differences between WT and ITGA5 KO. Data distribution was assumed to be normal but this was not formally tested. (F) Flow cytometry analysis of cell surface RUSH-α5 levels in adherent versus suspension U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP analyzed as in E are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test, data distribution was assumed to be normal but this was not formally tested. (G) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas) in WT and ITGA5 KO U2OS cells ± biotin treatment for the indicated times. One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal but this was not formally tested. Data are mean ± SD; N = 59 WT cells, 53 ITGA5 KO cells, pooled from three independent experiments. Source data are available for this figure: SourceData FS4.

Early release of RUSH-α5 is adhesion dependent and polarized recruitment to protrusions is supported by endogenous integrin α5. (A–D) Validation of ITGA5 CRISPR-Cas9 KO U2OS cells. (A) Western blot analysis of WT and ITGA5 KO single cell clones showing the efficiency of the CRISPR-Cas9 ITGA5 KO in U2OS cells. (B) Genome sequence alignment of U2OS WT and ITGA5 KO clones with the ITGA5 WT sequence. The targeted exon and the gRNA used for CRISPR KO positions are indicated. (C) Representative flow cytometry analysis of cell surface integrin α5 in U2OS WT and ITGA5 KO clones. (D) Images of WT and ITGA5 KO U2OS clones stained for active integrin α5 (SNAKA51) and paxillin. Scale bar: 20 µm. (E) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in WT and ITGA5 KO U2OS cells transfected with RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test showed no significant differences between WT and ITGA5 KO. Data distribution was assumed to be normal but this was not formally tested. (F) Flow cytometry analysis of cell surface RUSH-α5 levels in adherent versus suspension U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP analyzed as in E are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test, data distribution was assumed to be normal but this was not formally tested. (G) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas) in WT and ITGA5 KO U2OS cells ± biotin treatment for the indicated times. One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal but this was not formally tested. Data are mean ± SD; N = 59 WT cells, 53 ITGA5 KO cells, pooled from three independent experiments. Source data are available for this figure: SourceData FS4.

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