Journal: PloS one

Article Title: Inhibition of FOXO3 tumor suppressor function by betaTrCP1 through ubiquitin-mediated degradation in a tumor mouse model.

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: Figure 1. bTrCP1 protein interacts with FOXO3 protein and induces its ubiquitination in vivo and in vitro in an IKKb–phosphorylation dependent manner. (A) Endogenous proteins in total lysates of MCF-7 cells were subjected to IP with an antibody (Ab) as indicated followed by immunoblotting (IB) with an anti-bTrCP1 or anti-FOXO3 Ab. A rabbit IgG or an isotype mouse IgG were included as an IP negative control. The input IB data indicated the integrity of lysates used for IP. (B) 293T cells were cotransfected with HA-ubiquitin (Ub) and FOXO3-Myc or FOXO3-S644A expression vectors plus IKKb or a control vector (pCDNA3.1) as indicated. At 24 hours after transfection, cells were treated with either DMSO (control) or the IKKb inhibitor (2 mM) plus the proteasome inhibitor MG-132 (10 mM) for 5 hours. Total lysates were prepared and sonicated extensively for preventing non-covalent protein-protein associations, and subjected to IP with an anti-myc Ab and followed by IB analysis with an anti-HA Ab. The typical pattern of HA-tagged Ub was highlighted. The input IB data were performed with Abs against FOXO3, IKKb, phospho-IkBa (p-IkBa), IkBa, bTrCP1, and b-actin to show the expressions of transfected vectors in cells as indicated, the effect of the IKKb inhibitor (as indicated by the repression of p-IkBa level), and the integrity of each protein in total lysates used for IP. (C) IP was performed with the lysates of 293T cells that were cotransfected with the expression vectors as indicated, treated with MG-132 as described above, and followed by IB with an anti-HA Ab. The input IB data were performed with Abs against myc-tag, FOXO3, bTrCP1, IKKb, and b-actin to show the expressions of transfected vectors in cells and the integrity of lysates used for IP. (D) Firstly, 293T cells were transfected with either control-shRNA or bTrCP1-shRNA vectors. Twenty four hours post transfection, these cells were cotransfected with the expression vectors as indicated. Then, at 24 hours after cotransfection, cells were treated with MG-132 (20 mM) for 4 hours. The total lysates of these 293T cells were prepared and IP was performed as described above and followed by IB with an anti-HA Ab. The input IB data were performed with Abs against myc-tag, bTrCP1, IKKb, and b-actin as described. (E) 293T cells were cotransfected with various doses of bTrCP1 expression vector and mutant bTrCP1DF vector and other expression vectors as indicated. IP was performed with the lysates of these transfected 293T cells, treated with MG-132 as described above, and followed by IB with an anti-HA Ab. The input IB data were performed with Abs against FOXO3, IKKb, bTrCP1, and b-actin to show the expressions of transfected vectors and endogenous proteins in transfected cells and the integrity of lysates used for IP. (F) 293T cells were cotransfected with various doses of IKKb expression vector and other expression vectors as indicated. IP was performed with the lysates of these transfected 293T cells, treated with MG-132, and followed by IB with an anti-HA Ab as described above. The input IB data were performed with the indicated Abs as described above. (G) In vitro ubiquitination assays. The indicated target proteins GST-FO(1–300), GST-FO(301–673), and GST (negative control) were incubated with or without E3-ligase protein GST-bTrCP1 in Ub buffer containing E1, E2 (UbcH5b), Mg-ATP, biotinylated ubiquitin, and IKKb and analyzed by SDS-PAGE and IB with streptavidin conjugated with HRP or an anti-GST Ab as protein controls (lower panel). (H) The indicated target proteins were incubated with wild-type E3-ligase protein GST-bTrCP1 or mutant GST-bTrCP1DF in Ub buffer, and analyzed as described above. The positive signals are highlighted with *. doi:10.1371/journal.pone.0011171.g001

Article Snippet: The control vector (without shRNA), the scrambled shRNA control vector, and the bTrCP1-shRNAs vectors were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Ubiquitin Proteomics, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Negative Control, Expressing, Control, Plasmid Preparation, Transfection, Sonication, shRNA, Cotransfection, Mutagenesis, Incubation, SDS Page

Journal: PloS one

Article Title: Inhibition of FOXO3 tumor suppressor function by betaTrCP1 through ubiquitin-mediated degradation in a tumor mouse model.

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: Figure 4. DNA damage induces apoptosis in cells lacking bTrCP1 that can be reverted by silencing FOXO3 and ectopic expression of bTrCP1 promotes tumor growth whereas silencing bTrCP1 in cancer cells suppresses tumorigenesis in vivo. (A) DNA samples extracted from Wt MEF or bTrCP1(–/–) MEF cells treated with camptothecin (CPT) (20 mM) for 24 or 48 h or control (DMSO) were subjected to DNA fragmentation assay. Equal amounts of the extracted DNA (2 mg/lane) and size markers (M) were subjected to electrophoresis on 2% agarose gels, which were stained with ethidium bromide and photographed. (B) MEF cells treated with CPT (20 mM) or DMSO (control) (0 h) for 48 h were stained with propidium iodide (PI), and the cell-cycle profiles were determined by flow cytometry. The changes in percentage of cell-cycle statuses between CPT treatment and control were shown in a histogram. (C) BT549 cells were transfected with bTrCP1-siRNA (si-bTrCP1) or control RNA (si-control). DNA fragmentation assay was performed as described above. (D) BT549 cells were transfected with control RNA (si-control) alone or transfected with FOXO3-siRNA (si-FOXO3) plus bTrCP1-siRNA (si-bTrCP1). DNA fragmentation assay was performed. (E) The 231 cells were transfected with bTrCP1- siRNA (designated 231-bTrCP-kd) or transfected with a bTrCP1 expression vector (designated 231-bTrCP-ov). These transfected cells or 231 control cells were injected into the nude mice as described. *, P,0.05 between 231 control versus 231-bTrCP-kd or 231-bTrCP-ov. (F) At 28 days after tumor cell implantation, breast tumors derived from the nude mice bearing 231 (control) or 231-bTrCP-ov or -bTrCP-kd tumors were resected, fixed, sectioned, and placed on slides. Tumor specimens were subjected to immuno-histochemical staining with an Ab specific to bTrCP1 or FOXO3. Slides were examined at 406 magnification with a microscope and representative fields are shown. Scale bars indicate 50 mm. (G) Total lysates prepared from 231-bTrCP-ov and 231-bTrCP-kd breast tumor specimens were subjected to IB analysis with Abs against FOXO3, bTrCP1, and b-actin (loading control). (H) A diagram depicts the role of bTrCP1 in promoting tumorigenesis through inducing degradation of FOXO3 protein. The subcellular localization of FOXO3 is shown to denote FOXO3 activity. doi:10.1371/journal.pone.0011171.g004

Article Snippet: The control vector (without shRNA), the scrambled shRNA control vector, and the bTrCP1-shRNAs vectors were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Expressing, In Vivo, Control, DNA Fragmentation Assay, Electrophoresis, Staining, Flow Cytometry, Transfection, Plasmid Preparation, Injection, Derivative Assay, Microscopy, Activity Assay