Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: Requirement of IGF-1R for p53 accumulation and activation in response to etoposide in MEFs. (A) Etoposide-mediated apoptosis is reduced in MEFs upon transfection with GFP-p53DD. R + and R − MEFs were transfected with either an empty vector or GFP-p53DD and treated with etoposide for 48 h. Apoptotic cell death was measured as described in . Values are mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01. The expression and activity of GFP-p53DD were determined using antibodies against GFP and p21. (B) Detection of p53 activation in MEFs. R + and R − MEFs were cotransfected with p53bs-luc (wild-type responsive elements) or p53ms-luc (mutant responsive elements) and a plasmid expressing renilla that served as an internal control. After etoposide treatments, luciferase activity was measured. Relative luciferase activity is expressed as light units normalized for renilla luciferase activity. Data shown are from one out of three independent experiments with comparable results (mean ± SD). (C) Induction of p53 and its downstream targets Mdm2 and p21 after etoposide treatment in R + and R − MEFs. R + and R − MEFs were treated with the indicated doses of etoposide for 12 h, and whole cell lysates were analyzed by Western blot analysis with the indicated antibodies. (D) AG1024 treatment impairs etoposide-induced p53 induction and activation in R + but not R − MEFs. R + and R − MEFs were pretreated with AG1024 before etoposide treatment. Whole cell lysates were harvested and subjected to Western blot analysis with the indicated antibodies. (E) Dose course of apoptosis induced by ionomycin in R + and R − MEFs. MEFs were subjected to different doses of ionomycin and collected after 36 h, and apoptosis was analyzed by flow cytometry. Values are mean ± SD from three independent experiments.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Activity Assay, Mutagenesis, Control, Luciferase, Western Blot, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: IGF-1R inhibition increases p53 protein stability. (A) Measurement of p53 protein stability in etoposide-treated MEFs. R + and R − MEFs were treated with etoposide for 24 h before exposure to CHX. Extracts prepared at the indicated times after the addition of CHX were analyzed by Western blot analysis (top). The stability of p53 protein was quantified by ImageQuant software (bottom). p53 band density was normalized to actin density, and then expressed relative to the t = 0 controls and plotted on a semilogarithmic scale by a linear regression program against the times of CHX treatments. Each decreased unit of log 2 (band density) is equivalent to one half life. (B) Measurement of the p53 protein stability in unstressed MEFs. R + and R − MEFs were treated with CHX for the indicated times. Quantitation of the stability of p53 protein was performed as described in A. Values are mean ± SD from three independent experiments. (C) Lack of IGF-1R leads to enhanced p53 protein stability. MEFs were pulse labeled with [ 35 S]methionine/cysteine and chased as described in Materials and methods. p53 protein was immunoprecipitated and resolved by SDS-PAGE (left) and the amount of 35 S was quantified by PhosphorImaging (right). (D) Conjugation of ubiquitin to p53 protein is reduced upon IGF-1R loss. R + and R − MEFs were harvested for immunoprecipitation. Equal amounts of immunoprecipitated p53 proteins were subjected to Western blot analysis with antibodies against ubiquitin.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Inhibition, Western Blot, Software, Quantitation Assay, Labeling, Immunoprecipitation, SDS Page, Conjugation Assay, Ubiquitin Proteomics

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: IGF-1R inhibition down-regulates Mdm2 expression at the translational level. (A) Measurement of Mdm2 expression in MEFs. Cell lysates were harvested and subjected to Western blot analysis with the indicated antibodies. (B) Down-regulation of Mdm2 by IGF-1R inhibition is independent of p53. p53 +/+ and p53 −/− HCT116 cells were incubated with or without AG1024. Expression of Mdm2 protein was determined by immunoblotting with the indicated antibodies. (C) Measurement of mdm2 mRNA levels upon AG1024 treatment. After AG1024 treatment, total cellular RNA was prepared and levels of mdm2 transcripts were revealed by semiquantitative RT-PCR. (D) IGF-1R inhibition reduces translation of mdm2 mRNA in a p53-independent manner. After AG1024 treatments, HCT116 p53 +/+ and p53 −/− cells were pulse labeled and newly synthesized. Mdm2 was immunoprecipitated from cells and analyzed by PhosphorImaging (top). Analysis of total cellular proteins by SDS-PAGE showed equal amounts of loading (bottom). (E) AG1024 treatment does not affect Mdm2 protein stability. HCT116 p53 +/+ cells were treated as described in C, and then exposed to CHX for the indicated times. Quantitative analysis of Mdm2 stability was performed as described in . Values are mean ± SD from three independent experiements. (F) HCT116 p53 +/+ cells were pulse labeled and chased for the indicated periods of time, followed by immunoprecipitation. The amount of 35 S was quantified by PhosphorImaging.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Inhibition, Expressing, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction, Labeling, Synthesized, Immunoprecipitation, SDS Page

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: The 5′ UTR of p53 and mdm2 transcript mediates IGF-1R–dependent regulation of p53 and Mdm2 translation. (A) AG1024 specifically reduces translational levels of p53. [ 35 S]methionine/cysteine pulse-labeled p27, c-fos, and p53 were immunoprecipitated from SK-hep1 cells treated with or without AG1024 and analyzed by PhosphorImaging (bottom). Expression levels of p27, c-fos, and p53 proteins were monitored by immunoblotting (top). Adjustment of brightness and contrast ( 35 S-p27 panel) was performed with Photoshop 8.0 software. (B) Chimeric UTR-luc constructs carrying the 5′ and/or 3′ UTR of p53, mdm2, and c-fos mRNA. Each construct is labeled with the corresponding number at the right side. (C) AG1024 inhibits p53 and Mdm2 translation at the level of translation initiation; a schematic presentation of the FLAG-p53, FLAG-Mdm2, and FLAG–c-fos constructs containing the corresponding UTRs, CrPV IRES, and EGFP (left). SK-hep1 cells were transfected with the indicated expression vectors. Cells were treated with or without AG1024 and analyzed for protein expression by immunoblotting with antibodies against FLAG and GFP. gfp mRNA levels were determined by semiquantitative RT-PCR. (D and E) 5′ UTRs of the p53 and mdm2 transcript impose IGF-1R signaling–mediated translational regulation. SK-hep1 cells were transiently cotransfected with the chimeric UTR-luc constructs and pRL–SV40–renilla as described in Material and methods. After AG1024 treatment, luciferase activity was measured. Relative luciferase activity is expressed as light units normalized for renilla luciferase activity. Data are presented as mean ± SD of three independent experiments performed in quadruplicate. Levels of luciferase transcripts were revealed by semiquantitative RT-PCR. The number at the bottom of the panel is representative of the corresponding chimeric construct shown in B.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Labeling, Immunoprecipitation, Expressing, Western Blot, Software, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: Reduced translational synthesis of p53 in R − MEFs. (A) Measurement of p53 mRNA levels in MEFs by Northern blot analysis. p53 mRNA levels were detected by Northern blot analysis in R + and R − MEFs. gapdh levels were shown as loading controls. (B) Reduced translation of p53 mRNA in R − MEFs. p53 protein was immunoprecipitated from R + and R − MEFs labeled with [ 35 S]methionine/cysteine and analyzed as described in (top, lanes 3 and 4). An SDS-PAGE gel confirmed equal loading of total cellular proteins.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Northern Blot, Immunoprecipitation, Labeling, SDS Page

Journal: PLoS ONE

Article Title: Genome-Wide Screening Identified That miR-134 Acts as a Metastasis Suppressor by Targeting Integrin β1 in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0087665

Figure Lengend Snippet: A. Schematic diagram of the screening strategy. B. The results of the primary wound-healing screening. Each dot represents the inhibition index of an individual miRNA. The cutoff value was >40% (inhibition) or <−50% (promotion). C. The results of the second round of screening with administration of the proliferation inhibitor 2′, 3′- dideoxythymidine (DDT). D. Transwell migration assays for selected miRNAs (*p<0.05). SK-HEP-1 cells were plated into each well of a 12-well plate and transfected with individual miRNA mimics. The number of cells per field was counted after cells were placed into the top chamber of each insert for 5.5 hours. The results were normalized against data from cells transfected with the negative control. Differences between two groups were analyzed by the Mann-Whitney test.

Article Snippet: For the migration assay, 3×10 4 SK-HEP-1 cells or 5×10 4 Huh-7 cells were seeded in the top chamber of the insert (BD Biosciences, NJ, USA).

Techniques: Inhibition, Migration, Transfection, Negative Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Genome-Wide Screening Identified That miR-134 Acts as a Metastasis Suppressor by Targeting Integrin β1 in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0087665

Figure Lengend Snippet: A and B. Transwell migration ( A ) and invasion ( B ) assays for SK-HEP-1 and Huh-7 cells stably overexpressing miR-134 or negative control (lenti-vector). Representative images are shown with the quantification of five randomly selected fields. C. Transwell migration and invasion assays of Huh-7 cells were performed after transfection with the miR-134 inhibitor or negative control. Differences between two groups were analyzed by the Mann-Whitney test. D. H & E staining of liver isolated from mice that received orthotopic injections of miR-134-transduced or vector-infected LM3 cells at 8 weeks after transplantation. Intrahepatic nodules were counted and analyzed using the Mann-Whitney test (n = 10 mice per group).

Article Snippet: For the migration assay, 3×10 4 SK-HEP-1 cells or 5×10 4 Huh-7 cells were seeded in the top chamber of the insert (BD Biosciences, NJ, USA).

Techniques: Migration, Stable Transfection, Negative Control, Plasmid Preparation, Transfection, MANN-WHITNEY, Staining, Isolation, Infection, Transplantation Assay

Journal: PLoS ONE

Article Title: Genome-Wide Screening Identified That miR-134 Acts as a Metastasis Suppressor by Targeting Integrin β1 in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0087665

Figure Lengend Snippet: A. Schema of the candidate genes identified by four independent approaches. B. Putative binding sequences of ITGB1 for miR-134 in the human (Hsa), chimpanzee (Ptr), rhesus (Mml) and mouse (Mmu). The ITGB1 seed sequences are highlighted by ruby italic letters. C. Putative miR-134-binding sites in the wild-type ITGB1 3′-UTR (highlighted by ruby letters) or 3′-UTR segments containing mutant (highlighted by blue letters) binding sites are shown. D. Relative luciferase activity assays for constructs containing luciferase and wild-type or mutant ITGB1 3′-UTRs were performed after cotransfection with a miR-134 mimic or negative control. The Renilla luciferase vector was cotransfected as an internal control. Data were analyzed by one-way ANOVA (p<0.01), and differences between two groups were assessed by the Bonferroni post-test. E. The mRNA expression levels of ITGB1 in SK-HEP-1 and Huh-7 cells overexpressing miR-134 or negative control. Differences between two groups were analyzed by the Mann-Whitney test. F. The protein levels of ITGB1 in SK-HEP-1 and Huh-7 cells infected with miR-134 or control lentivirus. GAPDH served as an internal control. G. Protein levels of ITGB1 in Huh-7 cells after transfection with the miR-134 inhibitor or inhibitor control. GAPDH served as an internal control.

Article Snippet: For the migration assay, 3×10 4 SK-HEP-1 cells or 5×10 4 Huh-7 cells were seeded in the top chamber of the insert (BD Biosciences, NJ, USA).

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Construct, Cotransfection, Negative Control, Plasmid Preparation, Expressing, MANN-WHITNEY, Infection, Transfection

Journal: PLoS ONE

Article Title: Genome-Wide Screening Identified That miR-134 Acts as a Metastasis Suppressor by Targeting Integrin β1 in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0087665

Figure Lengend Snippet: A and B. Transwell migration assays of (A) SK-HEP-1 and (B) Huh-7 cells stably overexpressing ITGB1. Representative images are shown with the quantification of five randomly selected fields. The bars represent the SEM of three independent experiments. Differences between two groups were analyzed by the Mann-Whitney test. Western blot analysis revealed that the protein expression of ITGB1 increased after infection with the PLVX-ITGB1 (ORF) virus. C. Transwell migration and invasion assays for SK-HEP-1-vector or SK-HEP-1- miR-134 cells were performed after infection with ITGB1 (ORF) or the vector control lentivirus. Transwell migration and invasion data were analyzed by a one-way ANOVA (p<0.01, p<0.01 respectively); differences between two groups were assessed by the Bonferroni post-test. ITGB1 protein levels were determined by Western blot analysis. D. Transwell migration and invasion assays for Huh-7-vector or Huh-7-miR-134 cells were performed after infection with ITGB1 (ORF) or vector control lentivirus. ITGB1 protein levels were determined by Western blot analysis. Transwell migration and invasion data were analyzed by a one-way ANOVA (p<0.01, p<0.01 respectively); differences between two groups were assessed by the Bonferroni post-test.

Article Snippet: For the migration assay, 3×10 4 SK-HEP-1 cells or 5×10 4 Huh-7 cells were seeded in the top chamber of the insert (BD Biosciences, NJ, USA).

Techniques: Migration, Stable Transfection, MANN-WHITNEY, Western Blot, Expressing, Infection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Genome-Wide Screening Identified That miR-134 Acts as a Metastasis Suppressor by Targeting Integrin β1 in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0087665

Figure Lengend Snippet: A. and C. Ectopic expression of miR-134 disrupted the stress fiber network in SK-HEP-1 ( A ) and Huh-7 cells ( C ). Stress fibers (polymerized actin) and actin filaments were detected by phalloidin staining. DAPI staining was used to detect nuclei. Scale bar: 20 µm. B. and D. The protein levels of FAK phosphorylation and activity of Rho/Rac1/Cdc42 GTPases in SK-HEP-1 ( B ) and Huh-7 ( D ) cells. Samples for the Western blot were analyzed on a single gel. E. Cellular adhesion assays were performed in SK-HEP-1 and Huh-7 cells. The results are representative of at least three independent experiments. Differences between two groups were analyzed by the Mann-Whitney test. NC: negative control, FN: fibronectin.

Article Snippet: For the migration assay, 3×10 4 SK-HEP-1 cells or 5×10 4 Huh-7 cells were seeded in the top chamber of the insert (BD Biosciences, NJ, USA).

Techniques: Expressing, Staining, Activity Assay, Western Blot, MANN-WHITNEY, Negative Control

Journal: PLoS ONE

Article Title: Genome-Wide Screening Identified That miR-134 Acts as a Metastasis Suppressor by Targeting Integrin β1 in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0087665

Figure Lengend Snippet: A. Ectopic expression of miR-134 disrupted the stress fiber network in SK-HEP-1 cells, and this effect was reversed by ITGB1 transfection. Stress fiber formation was repressed by transfecting siRhoA into SK-HEP-1-ITGB1 cells. F-actin was used as the stress fiber marker. DAPI staining was used to detect nuclei. Scale bar: 20 µm. B. FAK protein phosphorylation and the protein levels of the Rho GTPases in SK-HEP-1-ITGB1 cells were analyzed after transfection with miR-134 and siRhoA. C. The cellular adhesion assay was performed in SK-HEP-1 cells. The results are representative of at least three independent experiments, and data were analyzed by a one-way ANOVA (p<0.05); differences between two groups were assessed by the Bonferroni post-test. NC: negative control, FN: fibronectin.

Article Snippet: For the migration assay, 3×10 4 SK-HEP-1 cells or 5×10 4 Huh-7 cells were seeded in the top chamber of the insert (BD Biosciences, NJ, USA).

Techniques: Expressing, Transfection, Marker, Staining, Cell Adhesion Assay, Negative Control