Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( 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-βTrCP1 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 IKKβ or a control vector (pCDNA3.1) as indicated. At 24 hours after transfection, cells were treated with either DMSO (control) or the IKKβ inhibitor (2 µM) plus the proteasome inhibitor MG-132 (10 µM) 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, IKKβ, phospho-IκBα (p-IκBα), IκBα, βTrCP1, and β-actin to show the expressions of transfected vectors in cells as indicated, the effect of the IKKβ inhibitor (as indicated by the repression of p-IκBα 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, βTrCP1, IKKβ, and β-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 βTrCP1-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 µM) 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, βTrCP1, IKKβ, and β-actin as described. ( E ) 293T cells were cotransfected with various doses of βTrCP1 expression vector and mutant βTrCP1ΔF 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, IKKβ, βTrCP1, and β-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 IKKβ 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-βTrCP1 in Ub buffer containing E1, E2 (UbcH5b), Mg-ATP, biotinylated ubiquitin, and IKKβ 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-βTrCP1 or mutant GST-βTrCP1ΔF in Ub buffer, and analyzed as described above. The positive signals are highlighted with *.

Article Snippet: The control vector (without shRNA), the scrambled shRNA control vector, and the βTrCP1-shRNAs vectors were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Western Blot, Negative Control, Expressing, Plasmid Preparation, Transfection, Sonication, shRNA, Cotransfection, Mutagenesis, In Vitro, Incubation, SDS Page

Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( A ) The stability of FOXO3 protein was assayed by cycloheximide (CHX) chase in 293T cells co-transfected with FOXO3-myc and βTrCP1 or a control vector (pCDNA3.1). Lysates of the transfected cells were prepared at 2, 4, 6, 8 hour (h) and 0 h (control) after addition of CHX, and subjected to immunoblotting (IB) with an anti-myc Ab. Graph shows results of densitometric analysis of 3 CHX chase experiments (mean ± SD) using 0 h (control) as 100%. ( B ) MCF-7 cells were transfected with small interfering RNA (siRNA) targeting βTrCP1 or control-siRNA (control). Total lysates of the transfected cells were subjected to IB analysis with an Ab against βTrCP1 or FOXO3 or β-actin (loading control) as indicated. ( C ) Total lysates of wild-type (Wt) and βTrCP1(–/–) MEF cells were subjected to IB analysis with an Ab against FOXO3 or βTrCP1 or β-actin (loading control). ( D ) βTrCP1(–/–) and βTrCP1(+/+) MEF cells were fixed, and the expression and subcellular localization of endogenous FOXO3 was detected using an anti-FOXO3 Ab and followed by an Alexa Fluor 546 (red) -conjugated secondary Ab, and analyzed with fluorescence microscopy. A fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) was used to visualize the nuclei. An average of ∼200 cells stained with anti-FOXO3 Ab were analyzed and a histogram shows the relative FOXO3 expression level in MEF cells. ( E and F ) Total lysates of 293T cells cotransfected with FRE-luc (firefly luciferase (luc) reporter containing FOXO-responsive elements), pRL-TK (renilla luc as a transfection control for normalization), βTrCP1, FOXO3 (A) or FOXO3-S644A (FO-S644A) (B), plus IKKβ or Akt-DD (an active Akt) as indicated and subjected to luc assays.

Article Snippet: The control vector (without shRNA), the scrambled shRNA control vector, and the βTrCP1-shRNAs vectors were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Western Blot, Small Interfering RNA, Expressing, Fluorescence, Microscopy, Staining, Luciferase

Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( A ) DNA samples extracted from Wt MEF or βTrCP1(–/–) MEF cells treated with camptothecin (CPT) (20 µM) for 24 or 48 h or control (DMSO) were subjected to DNA fragmentation assay. Equal amounts of the extracted DNA (2 µg/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 µM) 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 βTrCP1-siRNA (si-βTrCP1) 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 βTrCP1-siRNA (si-βTrCP1). DNA fragmentation assay was performed. ( E ) The 231 cells were transfected with βTrCP1-siRNA (designated 231-βTrCP-kd) or transfected with a βTrCP1 expression vector (designated 231-βTrCP-ov). These transfected cells or 231 control cells were injected into the nude mice as described. *, P<0.05 between 231 control versus 231-βTrCP-kd or 231-βTrCP-ov. ( F ) At 28 days after tumor cell implantation, breast tumors derived from the nude mice bearing 231 (control) or 231-βTrCP-ov or -βTrCP-kd tumors were resected, fixed, sectioned, and placed on slides. Tumor specimens were subjected to immuno-histochemical staining with an Ab specific to βTrCP1 or FOXO3. Slides were examined at 40× magnification with a microscope and representative fields are shown. Scale bars indicate 50 µm. ( G ) Total lysates prepared from 231-βTrCP-ov and 231-βTrCP-kd breast tumor specimens were subjected to IB analysis with Abs against FOXO3, βTrCP1, and β-actin (loading control). ( H ) A diagram depicts the role of βTrCP1 in promoting tumorigenesis through inducing degradation of FOXO3 protein. The subcellular localization of FOXO3 is shown to denote FOXO3 activity.

Article Snippet: The control vector (without shRNA), the scrambled shRNA control vector, and the βTrCP1-shRNAs vectors were purchased from OriGene Technologies, Inc. (Rockville, MD).

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

Journal: Immunity

Article Title: Oncolytic viruses engineered to enforce leptin expression reprogram tumor-infiltrating T cell metabolism and promote tumor clearance

doi: 10.1016/j.immuni.2019.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PCMV3-untagged Negative Control Vector , Sino Biological Inc. , Cat# CV011.

Techniques: Western Blot, Recombinant, Negative Control, Plasmid Preparation, Software

Journal: Immunity

Article Title: Oncolytic viruses engineered to enforce leptin expression reprogram tumor-infiltrating T cell metabolism and promote tumor clearance

doi: 10.1016/j.immuni.2019.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PCMV3-untagged Negative Control Vector , Sino Biological Inc. , Cat# CV011.

Techniques: Western Blot, Recombinant, Negative Control, Plasmid Preparation, Software