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The Hippo signaling pathway is involved in numerous biological events ranging from early development to organogenesis and when disrupted, impacts various human diseases including cancer. The Hippo pathway also interacts with and controls the activity of other signaling pathways such as the TGFβ/Smad pathway, in which Hippo pathway activity influences the subcellular localization of Smad transcription factors. Here, we describe techniques for examining crosstalk between Hippo and TGFβ signaling in polarizing mammary epithelial cells. In addition, we provide detailed methods for analyzing the subcellular localization of the Hippo pathway effectors, Taz and Yap using both in vitro cultured epithelial cells and in vivo in pregastrulation mouse embryos., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BMMCs) from the Gab2-deficient mice grew poorly in response to KitL. KitL-induced ERK MAP kinase and Akt activation were impaired in Gab2-deficient BMMCs. These data indicate that Gab2 is required for mast cell development and KitL/c-Kit signaling.

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of the IL-6-induced signals regulating growth and differentiation. In the M1 myeloleukemic cell line, Stat3 is a critical transcription factor causing repression of c-myc and c-myb genes, expression of junB and IRF1, growth arrest at G1, and subsequent macrophage differentiation. To understand the mechanisms by which Stat3 causes such effects, we searched for other Stat3-regulated genes possibly involved in growth arrest. We identified this inducible molecule as p19INK4D using a specific antibody. Both p19INK4D mRNA and protein were rapidly induced by IL-6 treatment without requiring de novo protein synthesis and the induction was fully suppressed by dominant-negative forms of Stat3. Thus both Stat3-regulated events, repressions of c-myc and c-myb and induction of p19INK4D, are likely to be involved in IL-6-induced growth arrest in M1 cells.