Journal:

Article Title: Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival

doi: 10.1128/MCB.24.6.2226-2236.2004

Figure Lengend Snippet: Targeted disruption of the mouse Tid1 gene. (A) Partial restriction maps of the Tid1 locus, targeting vector, targeted locus, and targeted locus after Cre-mediated Tid1 deletion. The first three exons are indicated by solid boxes. WT, wild type; TK, thymidine kinase. (B) Genotyping ES cell clones by Southern blot hybridization of genomic DNA (restriction digestion with XbaI enzyme) with the probe shown in panel A. The genotype of each clone is indicated. T, targeted. (C) Genotyping mice derived from the cross between Tid1-targeted mice and general deletor mice by Southern blot analysis as described for panel B. (D) Genotyping Tid1+/+ and Tid1+/− mice by PCR. The PCR primers used for genotyping are indicated by arrows and are depicted along with maps of the corresponding alleles. On the right are the patterns of electrophoresed PCR products for Tid1+/+ and Tid1+/− mice.

Article Snippet: Mouse genomic DNA containing the Tid1 gene was isolated by PCR-based screening of a bacterial artificial chromosome library derived from 129/SvJ mouse embryonic stem (ES) cells (Incyte, St. Louis, Mo.).

Techniques: Plasmid Preparation, Clone Assay, Southern Blot, Hybridization, Derivative Assay

Journal:

Article Title: Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival

doi: 10.1128/MCB.24.6.2226-2236.2004

Figure Lengend Snippet: Tid1−/− blastocysts were viable, hatched, and formed an inner cell mass and trophectoderm. (A) Blastocysts derived from Tid1+/− intercrosses were cultured in vitro for 3 days. In each outgrowth, the inner cell mass (ICM) and trophectoderm showed no differences. TG, trophoblast giant cell. (B) The genotype of each outgrowth was determined by PCR as described in the legend to Fig. ​Fig.1D.1D. The patterns of electrophoresed nested-PCR products are shown for ES cells with Tid1+/+, Tid1+/−, and Tid1−/− genotypes.

Article Snippet: Mouse genomic DNA containing the Tid1 gene was isolated by PCR-based screening of a bacterial artificial chromosome library derived from 129/SvJ mouse embryonic stem (ES) cells (Incyte, St. Louis, Mo.).

Techniques: Derivative Assay, Cell Culture, In Vitro, Nested PCR

Journal:

Article Title: Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival

doi: 10.1128/MCB.24.6.2226-2236.2004

Figure Lengend Snippet: Tid1 gene product is required for cell survival. (A) MEFs with Tid1+/+, Tid1+/−, Tid1f/+, Tid1f/−, and Tid1f/f genotypes were infected with Ad-Cre (Cre) (+) or uninfected (−) as indicated. After 6 days, total cellular proteins from these cells were collected and separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with an antibody against mouse Tid1. The recombinant Tid1L and Tid1S proteins generated from 293T cells transfected with expression plasmids encoding Tid1-L or Tid1-S (two splicing isoforms of Tid1) were used as controls. u.p., unprocessed; p., processed; n.s., nonspecific. (B) After infection with nothing (Mock), control adenovirus (control), or Ad-Cre (Cre) as indicated, the above-mentioned MEF cell lines were analyzed for growth and viability by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays at the indicated time intervals. OD560, optical density at 560 nm. The error bars indicate standard deviations. (C) Tid1f/+ and Tid1f/− MEFs were treated as for panel A. After 9 days, genomic DNAs were purified from these cells and separated by agarose gel electrophoresis (top). Some MEFs were also treated with 50 μM zVAD (a caspase 3 inhibitor) as indicated. The relative amounts of viable cells for these treated MEFs were also assessed by MTT assays (bottom). (D) Tid1f/+ and Tid1f/− MEFs were treated with 5 μg of anisomycin/ml (50 μM zVAD) as indicated for 18 h, and their genomic DNAs (top) and viabilities (bottom) were analyzed as for panel C. The MTT assays were performed in triplicate and repeated at least three times (the values are means plus standard deviations). (E) Tid1f/+ and Tid1f/− MEFs were infected with Ad-Cre and collected at the indicated time intervals. The cells were fixed and stained with propidium iodide (PI) and analyzed by flow cytometry.

Article Snippet: Mouse genomic DNA containing the Tid1 gene was isolated by PCR-based screening of a bacterial artificial chromosome library derived from 129/SvJ mouse embryonic stem (ES) cells (Incyte, St. Louis, Mo.).

Techniques: Infection, Polyacrylamide Gel Electrophoresis, Western Blot, Recombinant, Generated, Transfection, Expressing, Purification, Agarose Gel Electrophoresis, Staining, Flow Cytometry

Journal:

Article Title: Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival

doi: 10.1128/MCB.24.6.2226-2236.2004

Figure Lengend Snippet: DnaJ domain-dependent interaction between Tid1 and Hsp70 is critical for cell survival. (A) Total proteins were collected from Tid1f/− MEFs infected with recombinant adenoviruses encoding human Tid1-L, Tid1-S, or Tid1-S (H121Q) as indicated and analyzed by Western blotting with the anti-Tid1 antibody. (B) Tid1f/− MEFs were first infected with Ad-Cre or Ad-β-gal adenovirus, and after 2 days, the treated cells were infected with adenoviruses encoding human Tid1-L, Tid1-S, or Tid1-S (H121Q) as indicated. Nine days after the first infection, the number of viable cells for these MEFs was determined by MTT assay. OD560, optical density at 560 nm. The error bars indicate standard deviations. (C) Endogenous Tid1-L and Tid1-S proteins coimmunoprecipitated with mtHsp70. Total protein from MEFs was used for immunoprecipitation (I.P.) experiments with antibody specific for Tid1, mtHsp70, or Hsc70. The resulting immunocomplexes were analyzed by immunoblotting (I.B.) with antibody against Tid1, mtHsp70, or Hsc70, as indicated.

Article Snippet: Mouse genomic DNA containing the Tid1 gene was isolated by PCR-based screening of a bacterial artificial chromosome library derived from 129/SvJ mouse embryonic stem (ES) cells (Incyte, St. Louis, Mo.).

Techniques: Infection, Recombinant, Western Blot, MTT Assay, Immunoprecipitation

Journal:

Article Title: Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival

doi: 10.1128/MCB.24.6.2226-2236.2004

Figure Lengend Snippet: Tid1 and mtHsp70 proteins colocalize within mitochondria. (A) Immunofluorescence staining of Tid1 and mtHsp70 proteins in Tid1f/+ and Tid1f/− MEFs 5 days after treatment with Ad-Cre. (B) The same Ad-Cre-treated Tid1f/+ and Tid1f/− MEFs were first stained with Mitotracker and subsequently double stained with antibody against Tid1, mtHsp70, cytochrome c (Cyto. C), or AIF as indicated.

Article Snippet: Mouse genomic DNA containing the Tid1 gene was isolated by PCR-based screening of a bacterial artificial chromosome library derived from 129/SvJ mouse embryonic stem (ES) cells (Incyte, St. Louis, Mo.).

Techniques: Immunofluorescence, Staining

Journal:

Article Title: Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival

doi: 10.1128/MCB.24.6.2226-2236.2004

Figure Lengend Snippet: Decreased expression of BAG1 and MEK7 genes is partially responsible for cell death resulting from cellular Tid1 removal. (A) Tid1f/+ and Tid1f/− MEFs were infected (+) with Ad-Cre, and after 6 days, total RNAs from these MEFs were collected and analyzed by semiquantitative RT-PCR with the specific primer set for BAG1, MEK7, or GAPDH. The PCR products were separated through 4% agarose. (B) Total cellular extracts were prepared from these Ad-Cre-treated Tid1f/+ and Tid1f/− cells and analyzed by immunoblotting with anti-Tid1, anti-BAG1, anti-MEK7, or anti β-actin antibodies as indicated. n.s., nonspecific. (C) Tid1f/− MEFs were infected with Ad-Cre on day 1 and infected with human recombinant Tid1, BAG1, or MEK7 adenovirus on day 3. The number of viable cells on day 9 for each treated cell type was measured by MTT assay. The rescue rate for cells treated with the combination of Ad-Cre and Ad-Tid1 was set as 100%, and 0% was set for cells treated with Ad-Cre only. The error bars indicate standard deviations.

Article Snippet: Mouse genomic DNA containing the Tid1 gene was isolated by PCR-based screening of a bacterial artificial chromosome library derived from 129/SvJ mouse embryonic stem (ES) cells (Incyte, St. Louis, Mo.).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Recombinant, MTT Assay