CYRI-A and CYRI-B cooperatively regulate matrix attachment and surface expression of integrin α5β1 in A-673 cells. (A and B) Representative immunofluorescence images of A-673 cells comparing the spreading propensity between the control pLKO and the CYRI DBKO cells on fibronectin (A). Scale bar = 40 µm. Graph shows a time course of cell–matrix attachment for control pLKO (blue), the single KO (green and orange), and the DBKO cells. (C and D) Flow cytometry analysis (C) and quantification (D) of the surface expression of integrin α5β1 in A-673 cells between control pLKO and the DBKO cells (DBKO1 and DBKO2). (E and F) Representative Western blot of the total level of integrin α5β1 in DBKO cells compared with control pLKO or single KO cells (E). qPCR analysis for gene expression of integrin α5β1 in pLKO and the DBKO cells (F). (G and H) Immunofluorescence images of the surface level of integrin α5β1 between the control pLKO and DBKO cells (#1 and #2; G). Scale bar = 10 µm. The quantification is shown in H. Data from ≥10 cells per experiment in a total of three independent experiments. Each experiment is color-coded. (I and J) Effect of CYRI-A expression in DBKO cells on area and number of integrin adhesion sites. Data from ≥10 cells per experiment in a total of three independent experiments. Each experiment is color-coded. For all graphs, mean ± SD. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test.