Identification of Mst1 and GAPDH interaction sites.

<p><i>A,</i> schematic representation of Mst1 deletion mutants. <i>B,</i> flag-GAPDH expression vector in combination of either empty vector or expression vectors of Myc-Mst1 mutants were cotransfected into HEK293T cells. Extracted proteins were precipitated by anti-Myc antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP-conjugated anti-FLAG or HRP-conjugated anti-Myc antibody. Lysates were also immunoblotted with anti-Flag antibody to show the expression levels of Flag-GAPDH in HEK293T cells. <i>C,</i> schematic representation of GAPDH deletion mutants. <i>D,</i> Flag-Mst1 expression vector in combination of either empty vector or expression vectors of Myc-GAPDH mutants were cotransfected into HEK293T cells. Extracted proteins were precipitated by anti-Myc antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP-conjugated anti-FLAG or HRP-conjugated anti-Myc antibody. Lysates were also immunoblotted with anti-Flag antibody to show the expression of Flag-Mst1 in HEK293 cells.</p

Phosphorylation of GAPDH by Mst1.

<p>A, 0.1 µg active Mst1 was incubated with different amounts of recombinant GAPDH for 60 min in the presence of ³²P-ATP in an <i>in vitro</i> phosphorylation assay. B, 0.1 µg active Mst1 was incubated with 4 µg bacterially expressed recombinant GAPDH for different time points in the presence of ³²P-ATP in an <i>in vitro</i> phosphorylation assay. Phosphorylation was detected by autoradiography. The data are representatives of 4 independent experiments.</p

Knockdown of GAPDH attenuates Mst1 activation and cell apoptosis in response to hypoxia/reoxygenation.

<p><b>A</b>, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cells were treated with hypoxia for 12 hours and reoxygenation for 24 hours. Mst1 was then immunoprecipitated and its activity was determined by an in vitro kinase assay using histone H2B as a substrate. <b>B</b>, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cells were treated with hypoxia/reoxygenation and the cell apoptosis was determined by using the TUNEL staining kit (Roche). Values are means ± SEM obtained from 4 experiments.</p

Knockdown of GAPDH attenuates Mst1 activation and cell apoptosis in response to chelerythrine.

<p>A, Total RNA extracted from control siRNA (siCTL) and GAPDH siRNA transfected cells was analyzed for the expression of GAPDH in NRCMs by qRT-PCR. B, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cell lysates were then subjected to western blot analysis to detect the expression of GAPDH. C, NRVMs were transfected with either control siRNA or GAPDH siRNA. 72 hours after transfection, cells were treated with chelerythrine (5 µM) for 2 hours. Mst1 was then immunoprecipitated and its activity was determined by an in vitro kinase assay using histone H2B as a substrate. D, NRVMs were transfected with either control siRNA or GAPDH siRNA. 24 hours after siRNA transfection, cells were then transduced with either Ad-LacZ or Ad-Mst1 (MOI = 50). 48 hours after virus transduction, NRVMs were treated with chelerythrine (5 µM) for 2 hours and the cell apoptosis was determined by using the TUNEL staining kit (Roche). Values are means ± SEM obtained from 4 experiments.</p

Activation of Mst1 kinase by GAPDH.

<p>A, 0.1 µg active Mst1 was incubated with either 4 µg of recombinant GAPDH or MBP for 60 min in the presence of ³²P-ATP in an <i>in vitro</i> phosphorylation assay. Phosphorylation was detected by autoradiography. B, Mst1 immunoprecipitated from HEK293 cells transfected with either Myc-Mst1 or Myc-Mst1 plus Flag-GAPDH expression vectors was incubated with 2 µg MBP for the in vitro kinase assay. Kinase assay was carried out in the presence of ³²P-ATP for 60 min. Phosphorylation was detected by autoradiography. C, Phosphorylation levels of Mst1 and MBP were quantified by densitometry of autoradiograms. Values are means ± SEM obtained from 3 experiments.</p

Overexpression of GAPDH enhances Mst1 mediated cardiomyocyte apoptosis.

<p>A, NRVMs were transduced with either Ad-LacZ or Ad-GAPDH (MOI = 30). 48 hr after transduction, cell lysates were subjected to western blot analysis to detect the expression of GAPDH. B, NRVMs were transduced with Ad-LacZ or Ad-GAPDH at 30 mois. Twenty-four hours after transduction, myocytes were transduced with Ad-LacZ or Ad-Mst1 at 30 mois. 48 hours after the second transduction, cytoplasmic accumulation of mono- and oligonucleosomes was quantitated by the Cell Death Detection ELISA. Values are means ± SEM obtained from 4 experiments. C, NRVMs were transduced with Ad-LacZ, Ad-GAPDH, or Ad-DNMST with different combinations (total 60 mois). 48 hours after the transduction, the cells were treated with chelerythrine (5 µM) for 2 hours, the cytoplasmic accumulation of mono- and oligonucleosomes was then quantitated by the Cell Death Detection ELISA. Values are means ± SEM obtained from 4 experiments.</p

Association of Mst1 with GAPDH in cardiomyocytes.

<p>A. Cell lysates obtained from NRVMs were immunoprecipitated with anti-Mst1 antibody and then separated by 12% SDS-PAGE. Transferred membrane was immunoblotted with either anti-GAPDH or Mst1 antibody. B, Tissue homogenates obtained from normal mouse heart and hypertrophic heart were immunoprecipitated with anti-Mst1 antibody and then separated by 12% SDS-PAGE. Transferred membrane was immunoblotted with either anti-GAPDH or Mst1 antibody.</p

Physical interaction between Mst1 and GAPDH by immunoprecipitation analysis.

<p>A, Myc-Mst1 expression vector in combination of either empty vector or pFlag-GAPDH were co-transfected into HEK293 cells. Extracted proteins were precipitated by anti-FLAG antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP conjugated anti-Myc or HRP conjugated anti-FLAG antibody. B, Flag-GAPDH expression vector in combination of either empty vector, pMT2-Myc-Mst1 were co-transfected into HEK293 cells. Extracted proteins were precipitated by anti-Myc antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either HRP conjugated anti-Myc or HRP conjugated anti-FLAG antibody. C, HEK293 cells were transfected with Flag-Mst1. 48 hr after transfection, cells were then treated with etoposide (100 M) or TNF- (20 ng/ml) for 6 hrs. Extracted proteins were precipitated by anti-Flag antibody and then separated by 12% SDS-PAGE. The transferred membrane was immunoblotted with either anti-Mst1 or anti-Flag antibodies.</p

STAT5 phosphorylation in Stat5b transgenic mice.

<p>(A) Representative Western blotting analysis for phosphorylated and total STAT5 in F1 and NOD Stat5b transgenic lines. These mice did not have detectable signs of lymphoma based on physical examination and FACS analysis of T cell phenotypes. Protein extracts from thymus were used for the experiment. (B) Representative Western blotting analysis of phosphorylated STAT5 with thymus protein extracts from NOD.Stat5b transgenic mice with lymphoma. (C) STAT5 phosphorylation status in different cell types. Thymocytes and splenocytes from NOD Stat5b^Tg mice were analyzed by intracellular staining for phosphorylated STAT5 with an anti–pTyr694-STAT5 antibody. (D) FACS analysis showing progressive increase of STAT5 phosphorylation in thymocytes of NOD.Stat5b^Tg mice. Data for thymus are shown here for 6, 12 and 16 week old NOD.Stat5b^Tg mice (all without tumor). Representative data are shown from 1 of 3 similar experiments.</p

Phenotypes of CD8⁺ lymphomas and T cell development in NOD.Stat5b^Tg mice.

<p>(A) Three different types of lymphoma cells are illustrated. The vast majority of lymphomas (57/60) are similar to the Onco 2 mouse, e.g., with CD4⁺CD8⁺ double positive and CD8⁺ single positive thymocytes. Two mice with lymphomas had predominantly CD8⁺ single-positive thymocytes (Onco 18) and one mouse had predominantly CD4⁺CD8⁺ double-positive thymocytes (Onco 29). (B) Phenotypes of metastatic tumors. Tumor phenotypes are identical in different lymphoid organs including spleen (SP), thymus (Thy) and cervical lymph node (CN) (a, b, c). Tumor cells (5×10⁶) from thymus of the mouse as shown in b were subcutaneously injected at the back of regular NOD mice. Tumor formation was observed at the site of injection 10–20 days later and tumor cells also migrate to other lymphoid organs including spleen (d), thymus (e) and cervical node (f). Tumor phenotypes are identical at the injection site (g, dorsal) and other lymphoid organs. (C) CD25 expression in thymus of NOD.Stat5b^Tg mice and littermate controls at 6 weeks and lymphoma population in cervical node. (D) T cell phenotypes in the thymus (Thy) and spleen (Sp) of NOD.Stat5b^Tg mice and littermate controls at 6 weeks (6 w) and 12 weeks (12 w) of age. (E) T cell phenotypes in the thymus (Thy) and spleen (Sp) of NOD/B6 F1.Stat5b^Tg mice and littermate controls at 6 weeks (6 w) and 12 weeks (12 w) of age. (F) CD44 and CD122 expression of CD8⁺ T cells from spleens and thymi of NOD.Stat5b^Tg mice (16 weeks of age). Representative data are shown from 1 of 4 similar experiments.</p