The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states

Z-cad sensor loses GFP expression early and gains RFP expression later upon miR-200c induction. All collected time points for time-lapse microscopy (from Fig. 1c) of identical grids within cell culture plate are shown for each treatment group. A) –DOX control. B) 2 μg/mL DOX treatment to induce miR-200c. All time points after DOX treatment are indicated. Scale bars = 50 μm. (TIF 9490 kb

Additional file 1: Figure S1. of The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states

miR-200 sensor construct and validation; Z-cad sensor validation. A) Five miR-200 family binding sites were placed downstream of d2GFP in the FUGW lentiviral expression vector. B) Fluorescent microscopy of T11 cells containing the Z-cad dual sensor after 4 days of doxycycline treatment. C) Flow cytometry analysis of d2GFP-200 or control d2GFP expression and Ecad-RFP upon miR-200c/141 induction after 4 days of 2 μg/mL doxycycline treatment in the indicated cell lines (n = 3 biological replicates per group). D) Flow cytometry analysis of BLSL12 breast cancer cells containing the indicated sensors. miR-200c/141 was induced with 2 μg/mL doxycycline for 4 days. E) Fluorescent confocal microscopy of BLSL12 cells containing Z-cad dual sensor after 4 days of doxycycline treatment. Scale bar = 20 μm. (TIF 4770 kb

Additional file 6: Figure S6. of The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states

Z-cad population gates and their CD24/CD44 profiles. A) Flow cytometry analysis was used to gate HMLER cells based on the indicated Z-cad expression profiles (in red boxes). Gated cells from each population were subsequently analyzed for CD24 and CD44 expression as shown in B). (TIF 1218 kb

Mitochondrial dysfunction in NnaD mutant flies and Purkinje cell degeneration mice reveals a role for Nna proteins in neuronal bioenergetics

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronge d approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction

Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models

We have adapted the avian leukosis virus RCAS (replication-competent avian sarcoma-leukosis virus LTR splice acceptor)-mediated somatic gene transfer technique to introduce oncogenes into mammary cells in mice transgenic for the avian subgroup A receptor gene, tva, under control of the mouse mammary tumor virus (MMTV) promoter. Intraductal instillation of an RCAS vector carrying the polyoma middle T antigen (PyMT) gene (RCAS-PyMT) induced multiple, oligoclonal tumors within 3 weeks in infected mammary glands of MMTV-tva transgenic mice. The rapid appearance of these tumors from a relatively small pool of infected cells (estimated to be ≈2 × 10(3) cells per gland by infection with RCAS carrying a GFP gene; RCAS-GFP) was accompanied by a high fraction of cells positive for Ki67, Cyclin D1, and c-Myc, implying strong proliferation competence. Furthermore, the tumors displayed greater cellular heterogeneity than did tumors arising in MMTV-PyMT mice, suggesting that RCAS-PyMT transforms a relatively immature cell type. Infection of mice transgenic for both MMTV-Wnt-1 and MMTV-tva with RCAS virus carrying an activated Neu oncogene dramatically enhanced tumor formation over what is observed in uninfected bitransgenic animals. We conclude that infection of mammary glands with retrovirus vectors is an efficient means to screen candidate oncogenes for their capacity to initiate or promote mammary carcinogenesis in the mouse