Journal: Stem Cells (Dayton, Ohio)

Article Title: A histone deacetylase 7‐derived peptide promotes vascular regeneration via facilitating 14‐3‐3γ phosphorylation

doi: 10.1002/stem.3122

Figure Lengend Snippet: The MEKK1, 7A, and 14‐3‐3γ formed a novel signaling pathway downstream vascular endothelial growth factor (VEGF). A, Knockdown of Mekk1 abolished VEGF‐induced 14‐3‐3γ phosphorylation in differentiated Sca1 + ‐VPCs. The Sca1 + ‐VPCs were transfected with Mekk1 siRNA ( Mekk1si ) and cultured for 3 days, then treated with 5 ng/mL of VEGF in serum‐free medium for 30 minutes, followed by Western blotting with anti‐phospho‐MEKK1Ser393 (pMEKK1), anti‐MEKK1, anti‐phospho‐14‐3‐3γThr145 (p14‐3‐3γ), and anti‐14‐3‐3γ antibodies. Control siRNA ( ctlsi ) and GAPDH were included as siRNA and loading controls. pM/M/G: pMEKK1/MEKK1/ GAPDH; p14/14/G: p14‐3‐3γ/14‐3‐3γ/GAPDH. B, The 7Ap‐peptide induced 14‐3‐3γ phosphorylation independent of MEKK1. The Mekk1 knockdown Sca1 + ‐VPCs were pretreated with 1 ng/mL peptides for 1 hour and then treated with 5 ng/mL VEGF for 30 minutes, followed by Western blotting with anti‐phospho‐14‐3‐3γThr145(p14‐3‐3γ) and anti‐14‐3‐3γ antibodies. Phosphate‐buffered saline and 1% bovine serum albumin were included as vehicle controls for peptide and VEGF, respectively. GAPDH was included as a loading control. C, 7A deficiency abolished VEGF‐induced 14‐3‐3γ phosphorylation. The Sca1 + ‐VPCs were isolated from wild‐type (WT), heterozygous (HZ), and homozygous (KO) mice arteries and treated with 5 ng/mL VEGF for 30 minutes, followed by Western blot analysis of 14‐3‐3γ phosphorylation. 14‐3‐3γ and GAPDH were included as loading controls. Note that HDAC7 expression was not affected by the transgenic handling. D, Local delivery of synthetic 7A‐peptide rescued 14‐3‐3γ phosphorylation in KO mice. Double immunofluorescence staining with anti‐Sca1 (red) and anti‐p14‐3‐3γT145 (green) antibodies was performed on the sections isolated from the femoral artery injured WT and KO mice 3 days postsurgery. In KO + 7A group, 200 μL of Pluronic F‐127 gel containing 10 ng/mL 7A was applied to the injury sites. Five mice were used for each group. The data presented are representative images or mean values of three independent experiments. For statistical analysis, the two‐way analysis of variance followed by Tukey's multiple comparisons test was used * P < .05. VPC, vascular progenitor cell

Article Snippet: The antibody against GAPDH (sc‐25 778), HDAC7(sc‐74 563), and the siRNAs (control siRNA [sc‐37007], MEKK1 siRNA [sc‐35899], and 14‐3‐3γ siRNA [sc‐29 584]) were purchased from Santa Cruz Biotechnology (Dallas, Texas).

Techniques: Knockdown, Phospho-proteomics, Transfection, Cell Culture, Western Blot, Control, Saline, Isolation, Expressing, Transgenic Assay, Double Immunofluorescence Staining

Journal: Molecular and Cellular Biology

Article Title: ΔNp73 Facilitates Cell Immortalization and Cooperates with Oncogenic Ras in Cellular Transformation In Vivo

doi: 10.1128/mcb.23.16.5540-5555.2003

Figure Lengend Snippet: FIG. 5. Np73 cooperates with cMyc and E1A in cell proliferation and colony formation. (A) Morphology of 129 MEFs infected with retroviruses expressing GFP only, Np73 only, cMyc only, or cMyc together with wild-type or mutant Np73. mtNp73 stands for Np73 L322P. (B) Colony formation assay of MEFs transduced with the indicated plasmids. The bar graph shows the quantitation of three independent experiments plus the standard deviation. (C) Short-term growth curve of MEFs infected with retroviruses coexpressing either E1A and GFP or E1A and Np73.

Article Snippet: Whole-cell lysates (50 g of protein) were separated in sodium dodecyl sulfate–10% acrylamide gels, blotted onto a Protran BA83 nitrocellulose membrane (Schleicher & Schuell), and then incubated with antibodies specific for p73 (ER15; Oncogene Sciences); p53 (CM-5; Vector Laboratories); p21 (F5), p16 (M156), pRb (IF8), cMyc (N-262), and E1A (135-5) (all from Santa Cruz Biochemicals); Ras (R02120; Transduction Laboratories); p19ARF (Ab80; Novus Biologicals); and Erk (3A7; Cell Signaling) to detect the respective proteins.

Techniques: Infection, Expressing, Mutagenesis, Colony Assay, Transduction, Quantitation Assay, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: ΔNp73 Facilitates Cell Immortalization and Cooperates with Oncogenic Ras in Cellular Transformation In Vivo

doi: 10.1128/mcb.23.16.5540-5555.2003

Figure Lengend Snippet: FIG. 6. Np73 compromises p53-dependent apoptosis in primary fibroblasts. (A) 129 wild-type MEFs were infected with the indicated retroviruses 2 days prior to treatment with camptothecin (5 M for 10 h). (B) 129 wild-type MEFs, p53/ MEFs, or p73/ MEFs were infected with the indicated retroviruses 2 days prior to treatment with etoposide (5 M for 10 h). (A and B) Quantitation of cell death in expressing cells by TUNEL assays. Np73 expression in E1A- or cMyc-sensitized MEFs decreased DNA damage-triggered, p53-mediated apoptosis. The results represent three independent experiments plus the standard deviation. mtNp73 stands for Np73 L322P; dnp53 stands for p53 R175H. (C) Representative fields of TUNEL assays from panel B.

Article Snippet: Whole-cell lysates (50 g of protein) were separated in sodium dodecyl sulfate–10% acrylamide gels, blotted onto a Protran BA83 nitrocellulose membrane (Schleicher & Schuell), and then incubated with antibodies specific for p73 (ER15; Oncogene Sciences); p53 (CM-5; Vector Laboratories); p21 (F5), p16 (M156), pRb (IF8), cMyc (N-262), and E1A (135-5) (all from Santa Cruz Biochemicals); Ras (R02120; Transduction Laboratories); p19ARF (Ab80; Novus Biologicals); and Erk (3A7; Cell Signaling) to detect the respective proteins.

Techniques: Infection, Quantitation Assay, Expressing, TUNEL Assay, Standard Deviation