Figure 2.

CORO7 is critical for the activation of the Hippo pathway in mammalian cells.A, MDA-MB-231 cells were transfected with either nontargeting control siRNA (si-control) or siRNA targeting CORO7 (si-CORO7) and were deprived of serum. The lysate samples were immunoblotted with anti-pYAP, anti-YAP, anti-CORO7, and antitubulin antibodies. B, equal numbers of MDA-MB-231 cells with (si-CORO7) or without (si-control) CORO7 knockdown were seeded in 1.9, 3.8, 9.6, and 21.5 cm² culture plates to achieve different cell densities 24  h before collecting, and the lysate samples were immunoblotted with the same antibodies as in (A). C, MDA-MB-231 cells were transfected with either nontargeting control siRNA (si-control) or siRNA targeting CORO7 (si-CORO7) and were treated with LatB (0.25 μg/ml) to cause cytoskeleton damage. The cell lysate samples were immunoblotted with the same antibodies as in (A). Normalized signal intensities of pYAP bands relative to the total YAP protein levels were represented as numbers in (A–C). D, HEK293T cells were treated with either nontargeting control siRNA (si-control) or siRNA targeting CORO7 (si-CORO7) and were serum starved for 1 h (FBS). The cells were immunostained with anti-YAP antibody (red). Nuclei of the cells were stained with Hoechst 33342 (blue). The scale bar represents 10 μm. E, HEK293T cells with (si-CORO7) or without (si-control) CORO7 knockdown were cultured under a dense monolayer condition. The cells were immunostained with anti-YAP antibody. Nuclei of the cells were stained with Hoechst 33342 (blue). The scale bar represents 10 μm. F and G, wild-type (MDA-MB-231) or CORO7-lacking (CORO7 KO) MDA-MB-231 cells were cultured under serum deprivation (F) or LatB treatment (0.25 μg/ml) (G). The lysate samples were immunoblotted with the same antibodies as in (A). Nonspecific band is indicated with an asterisk in (G). Normalized signal intensities of pYAP bands relative to the total YAP protein levels were represented as numbers. H, wild-type (MDA-MB-231) or CORO7 KO MDA-MB-231 cells were cultured at high density as in (B) (lanes 4 and 8). mRNA expression levels of CTGF and CYR61 were determined by quantitative real-time PCR and normalized by a control gene, ACTB. The error bars represent ±S.D. from five independent experiments. Student's two-tailed t test was applied (∗∗∗p < 0.001). I, wild-type (MDA-MB-231) or CORO7 KO MDA-MB-231 cells were cultured at high density as in (H) and cotransfected with pRL-TK-encoding Renilla luciferase and 8× TBS-encoding firefly luciferase. About 24 h after the transfection, the cells were collected and subjected to dual-luciferase reporter assay (n = 3). Student's two-tailed t test was applied (∗∗∗p < 0.001). All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBS, fetal bovine serum; KO, knockout; LatB, latrunculin B; YAP, yes-associated protein.