Fig 1: Tyrosine kinase Src is a potential regulator of CORO7.A, representative pictures of the wings expressing RNAi targeting Src64B (Src64BRNAi) with or without expressing Pod1 or RNAi targeting pod1 (pod1RNAi) by MS1096-GAL4 as indicated. The scale bar represents 500 μm. B, statistical analysis of the wing size of each genotype in (A). The error bars represent ±S.D. from n > 20. One-way ANOVA and Tukey post-test were applied (∗p < 0.05; ∗∗∗p < 0.001). C, siRNA targeting CORO7 (si-CORO7) or nontargeting control siRNA (si-control) was transfected in MDA-MB-231 cells, and 50 nM dasatinib was treated for 1 h as indicated. The cell lysate samples were immunoblotted with anti-pYAP, anti-YAP, antitubulin, and anti-CORO7 antibodies. Quantification of the fold ratio of pS127-YAP bands to YAP protein levels under each indicated condition (right). The error bars represent ±S.D. from n = 3. One-way ANOVA and Tukey post-test were applied (∗∗p < 0.01). D, HEK293T cells expressing Flag-CORO7 and HA-Src were treated with 250 nM dasatinib for 2 h. The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-pTyr and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels. E, HEK293T cells were transfected with indicated mutant forms of Flag-CORO7 and a constitutively active form of Src (HA-Src Y530F). CORO7 3YF indicates that Tyr712, Tyr738, and Tyr758 were all mutated to phenylalanine. The lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-pTyr and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels. CORO7, coronin 7; YAP, yes-associated protein; HA, hemagglutinin; WCL, whole cell lysate.
Fig 2: Drosophila pod1 genetically interacts with Hippo pathway genes.A, schematic diagram of the genetic screen for Hippo pathway regulators. B, representative pictures of the wings of Pod1 overexpression (Pod1), pod1 knockdown (pod1RNAi), pod1 knockout (pod1KO) without or with Hpo knockdown (+HpoRNAi) or Yki:GFP (+Yki) overexpression by MS1096-GAL4. The PCV is indicated. The scale bar represents 500 μm. C, statistical analysis of the wing size of each genotype in (B). The error bars represent ± S.D. from n > 20. One-way ANOVA and Tukey post-test were applied (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). D, posterior regions of eye discs in Drosophila larvae. Yki:GFP, YkiS168A:GFP, Scalloped, and pod1RNAi were expressed by GMR-GAL4 as indicated. The scale bar represents 10 μm. GMR, glass multimer reporter; PCV, posterior cross vein.
Fig 3: CORO7 forms a complex with the components of the Hippo pathway.A–C, schematic representation of domains and truncated constructs of CORO7 (A), LATS1 (B), and SAV1 (C) used in coimmunoprecipitation assays. D–F, HA-LATS1 (D), HA-MST2 (E), or HA-SAV1 (F) was expressed together with wild-type (WT), N-terminal (N), or C-terminal (C) Flag-CORO7 in HEK293T cells. The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The whole cell lysate (WCL) samples were loaded for indicating the expression levels. G, HA-CORO7 was transfected with the Flag-tagged truncated forms of LATS1 in HEK293T cells, and the lysates were immunoprecipitated by anti-Flag antibody. Subsequently, they were immunoblotted with the same antibodies as in (D–F). The WCL samples were loaded for indicating the expression levels. H, Flag-CORO7 was transfected with the HA-tagged truncated forms of SAV1 in HEK293T cells. The lysates were immunoprecipitated by anti-HA antibody and immunoblotted with the same antibodies as in (D–F). The WCL samples were loaded for indicating the expression levels. I, HEK293T cells were transfected with HA-tagged truncated forms of SAV1, HA-MST2, and Flag-CORO7. The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with the same antibodies as in (D–F). The WCL samples were loaded for indicating the expression levels. Data information: Flag-tagged vector and HA-tagged vector were transfected as negative controls (−). All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBM, FERM-binding motif; HA, hemagglutinin; LATS1, large tumor suppressor kinase 1/2; MOB1, MOB kinase activator 1; MST2, mammalian sterile 20-like kinase 2; SARAH, Sav/Rassf/Hpo; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate.
Fig 4: Signals activating the Hippo pathway regulate complex formation between CORO7 and the components of the pathway.A, 24 h after seeded to 15% confluency, HEK293T cells transfected with HA-MST2, HA-SAV1, and Flag-CORO7 were deprived of serum (FBS) or treated with 0.25 μg/ml LatB for 1 h. The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels. The WCL samples were also immunoblotted with anti-pYAP antibody. B, HEK293T cells were transfected with Flag-CORO7, Myc-SAV1, and HA-NF2. Three different levels of HA-NF2 were expressed. The cell lysate samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA, anti-Flag, and anti-Myc antibodies. The WCL samples were loaded for indicating the expression levels. C, HEK293T cells expressing Myc-CORO7, HA-NF2, and Flag-SAV1 were deprived of serum (FBS). The cell lysate samples were immunoprecipitated with anti-Flag antibody and immunoblotted with the same antibodies as in (B). The WCL samples were loaded for indicating the expression levels. All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; FBS, fetal bovine serum; HA, hemagglutinin; LatB, latrunculin B; MST2, mammalian sterile 20-like kinase 2; NF2, neurofibromatosis type II; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate; YAP, yes-associated protein.
Fig 5: CORO7 is necessary for the formation of the core Hippo kinase complex.A, Flag-MST2, HA-LATS1, HA-SAV1, and HA-MOB1 were transfected in HEK293T cells to observe the coimmunoprecipitation between Flag-MST2 and the rest. siRNA targeting CORO7 was treated in the indicated lane (si-CORO7). The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The whole cell lysate (WCL) samples were loaded for indicating the expression levels, and CORO7 was immunoblotted to verify the efficiency of knockdown. B, flag-LATS1, HA-MST2, HA-SAV1, and HA-MOB1 were transfected in HEK293T cells to observe the coimmunoprecipitation between Flag-LATS1 and the rest. siRNA targeting CORO7 was treated in the indicated lane (si-CORO7). The lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-HA and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels, and CORO7 was immunoblotted to verify the efficiency of knockdown. C, flag-MST2 was expressed in HEK293T cells, and CORO7 was knocked down in the indicated lane (si-CORO7). Cells were deprived of FBS for 24 h, and the lysates were immunoprecipitated by anti-Flag antibody and immunoblotted with anti-LATS1, anti-SAV1, and anti-Flag antibodies. The WCL samples were loaded for indicating the expression levels, and CORO7 was immunoblotted to verify the efficiency of knockdown. All immunoblots are representative of at least three independent experiments. CORO7, coronin 7; HA, hemagglutinin; LATS1, large tumor suppressor kinase 1/2; MOB1, MOB kinase activator 1; MST2, mammalian sterile 20-like kinase 2; SAV1, salvador family WW domain–containing protein 1; WCL, whole cell lysate.
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