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